Food Chemistry 134 (2012) 445452

Contents lists available at SciVerse ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Characterisation and quantication of xanthones from the aril and pericarp of mangosteens (Garcinia mangostana L.) and a mangosteen containing functional beverage by HPLCDADMSn
Judith Wittenauer a,b, Susanne Falk b, Ute Schweiggert-Weisz a,, Reinhold Carle b
a b

Fraunhofer Institute for Process Engineering and Packaging, Department of Process Engineering, Giggenhauser Str. 35, D-85354 Freising, Germany Hohenheim University, Institute of Food Science and Biotechnology, Chair Plant Foodstuff Technology, Garbenstrae 25, D-70599 Stuttgart, Germany

a r t i c l e

i n f o

a b s t r a c t
Attention on mangosteen fruits as an ingredient of functional products is growing, particularly due to their rich content of xanthones. Whereas mangosteen products containing puree from the entire fruit of Garcinia mangostana L. are considered as novel food in the European Union, such products are widely used in the US due to their high antioxidant potential and traditional consumption in their countries of origin. With special emphasise on the xanthone prole and content, mangosteen pericarp, aril segments and a functional beverage made from whole mangosteens were compared. The fruit parts and the product showed a consistent pattern composed of mainly 7 xanthones, which could be unambiguously identied by LCMS. Based on collision-induced dissociation experiments, fragmentation pathways of xanthones were suggested. The quantication of 7 derivatives contained in the arils, the pericarp and the functional beverage allowed an estimation of the amounts of bioactives which are ingested by the consumption of fresh mangosteen fruits and beverages produced thereof. Total xanthone content of the pericarp was the highest, revealing its potential as functional ingredient followed by the aril segments and the functional beverage. It has been shown, that the content of bioactive xanthones in 90 mL of the beverage (i.e. the recommended daily dose) corresponds to about 0.9 g of pericarp and the aril segments (30 g) of a single mangosteen. Since residual parts of pericarp are always ingested after usual peeling of the fruit, xanthone concentrations exceeding those of the nutritional beverage have been ingested, thus allowing to establish a safe history of use. 2012 Elsevier Ltd. All rights reserved.

Article history: Received 20 April 2011 Received in revised form 19 January 2012 Accepted 16 February 2012 Available online 25 February 2012 Keywords: Xanthones Garcinia mangostana L. Aril Pericarp HPLCDADMSn

1. Introduction Garcinia mangostana L. (Clusiaceae), commonly known as mangosteen, is a tropical evergreen tree native to Malaysia. It bears dark purple or reddish fruits with a juicy pericarp containing 8 10 aril segments of different widths. In the ripening stage, mangosteen pericarp can be easily torn open manually at the equator revealing the edible aril segments (Palapol et al., 2009). They are white, soft and juicy with a sweet, slightly acidic taste and a kernel inside. Because of their pleasant aroma, mangosteens are regarded by many as one of the most relishable fruits. Although mangosteens are climacteric fruits that are picked from the tree when they are ready and continue to ripen, an import of the fruits proves to be difcult due to their perishable and fragile nature. Mangosteens show a limited shelf life with a beginning decay 14 days after picking. Additionally, the ripe fruits are very sensitive towards impact damage (Kanchanapoom & Kanchanapoom, 1998). Common meth Corresponding author. Tel.: +49 (0) 8161 491 431; fax: +49 (0) 8161 491 444.
E-mail address: ute.weisz@ivv.fraunhofer.de (U. Schweiggert-Weisz). 0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.02.094

ods to increase the storability and postharvest life of fruits like cool temperature and reduced concentrations of oxygen are not tolerated by mangosteens. Under such conditions, mangosteens suffer from quality loss, commonly known as chilling injury. Several studies have shown, that storage of mangosteens at temperatures below 12 C or an mechanical damage of the fruits induces lignin biosynthesis and lowered polyphenol content in the pericarp (Bunsiri, Ketsa, & Paull, 2003; Dangcham, Bowen, Ferguson, & Ketsa, 2008; Ketsa & Koolpluksee, 1993). Consequently, mangosteen pericarp hardens to a leather-like consistence, and the purple colour of the fruits turns into brown (Ketsa & Atantee, 1998; Palapol et al., 2009). Therefore, to prevent hardening of the pericarp, processing of mangosteens is mostly realised in their cultivation areas. The traditional use of mangosteens is manifold. They are mainly eaten fresh as dessert. However, the production of wine, preserves, jam and puree from the arils and the whole fruit are further traditional food applications (Bin Osman & Milan, 2006). A new potential market for purees made from the whole fruit is the production of functional beverages, mostly combined with constituents of other fruits. Since mangosteen pericarp and the whole fruit have

446

J. Wittenauer et al. / Food Chemistry 134 (2012) 445452

been used in Southeast Asia for centuries in the treatment of several diseases such as diarrhoea, dysentery, infections of the skin, mycosis, inammation, cholera and fever, manifold medicinal indications are attributed to the fruits (Obolskiy, Pischel, Siriwatanametanon, & Heinrich, 2009; Pedraza-Chaverri, CrdenasRodrguez, Orozco-Ibarra, & Prez-Rojas, 2008). Hence, mangosteen fruits rank among the most potent so-called super fruits, thus being increasingly used as active constituents in functional products. Owing to their traditional use in folk medicine, numerous studies have been performed to substantiate the putative health effects of mangosteens. Particular emphasis has been put on their rich content of prenylated and oxygenated xanthones, a class of polyphenols which is only synthesised within a small group of higher plants, fungi and lichens. Biological activities of these secondary plant metabolites, including their C-glycosides which occur in mango peels (Schieber, Berardini, & Carle, 2003), are due to the tricyclic xanthen-9-one core and their varying substitution pattern depending on the nature and position of substituents (Bennett & Lee, 1989; Peres, Nagem, & de Oliveira, 2000). The results of most studies indicate that xanthone extracts from G. mangostana L. display a high antioxidative capacity (Jung, Su, Keller, Mehta, & Kinghorn, 2006; Weecharangsan et al., 2006). Additionally, extensive literature has been reviewed concerning other potential biological activities (Obolskiy et al., 2009; Pedraza-Chaverri et al., 2008). Along with anti-inammatory, antibacterial, antifungal, antiviral and antiallergic properties, extracts and xanthones from mangosteens even showed antitumoral properties. Furthermore, many investigations have been performed to study the extraction and elucidate the structure of xanthones from mangosteens (Chaivisuthangkura et al., 2009; Jung et al., 2006; Walker, 2007; Zarena & Sankar, 2009). The majority of these studies focus on xanthones from the pericarp of mangosteens, where a-mangostin and c-mangostin were found to be the major constituents (Jung et al., 2006; Walker, 2007). The occurrence of mangostins in other parts of the mangosteen tree or fruit like aril segments (Mahabusarakam & Wiriyachitra, 1987), heartwood (Nilar, 2002), stem (Ee, Daud, Tauq-Yap, Ismail, & Rahmani, 2006) and seed (Suksamrarn et al., 2003) has only been reported by few authors. Beyond that, there is a general lack of clarity in literature dening the specic parts of G. mangostana fruits that were investigated. Considering the traditional use of the whole ripe fruits in the producing countries for many mangosteen products and their increasing application in functional beverages, it is surprising that studies on the xanthone content in pulp and processed products are rather limited (Ji, Avula, & Khan, 2007). Therefore, data about the composition in particular of bioactive xanthones in different parts of the fruit should be made available. By providing evidence of similar xanthone patterns of the pericarp and aril segments, health claims associated with the rind may be extended to the pulp originating from the arillus. Therefore, the objective of this work was the proling of prenylated and oxygenated xanthones from extracts of mangosteen pericarp as compared to aril segments and a functional beverage using HPLCDADMSn. Attended by the quantication of major xanthone species in pericarp, aril segments and the functional beverage, these data may contribute to an adequate classication of mangosteen products.

Thai mangosteens (G. mangostana L.) were obtained from Tropical Food Europe (Hofheim/Wallau, Germany) and the functional beverage was obtained from XanGo (Utah, USA). 2.2. Sample preparation Calyxes and stipes were removed with a stainless steel knife and fruits were carefully separated into pericarp and aril segments to enable precise differentiation of aril and pericarp constituents. Prior to analysis, pericarp and aril segments were reduced to small pieces and immediately lyophilised. The determination of xanthones was carried out according to a method of Schieber et al. (2003) with slight modications of the extraction procedure. Lyophilised pericarp and pulp were nely ground using a Grindomix GM 200 knife mill (Retsch, Haan, Germany) and passed through a 0.8 mm analytical sieve (Retsch, Haan, Germany). Aliquots of 0.4 g (pericarp) and 0.5 g (arils) of the powdered samples and 0.5 g ascorbic acid were weighted into Erlenmeyer asks, ushed with nitrogen and extracted with 50 mL methylene chloride for 45 min under shaking, using a KS 500 laboratory shaker (Janke & Kunkel, IKA Labortechnik, Staufen, Germany). The mixture was ltrated through a lter paper (Munktell, grade 3 hw), and the residue was extracted with 50 mL methylene chloride once again for 45 min to ensure exhaustive extraction. Supernatants were combined and the organic solvent was removed by evaporation in vacuo at 30 C. The residue was dissolved in 3 mL of methanol. After membrane-ltration (0.45 lm, Whatman, Dassel, Germany), the samples were directly used for the quantication of individual xanthones by HPLCDAD. Prior to the identication of individual compounds using LC MS, the crude extracts were puried by solid-phase extraction. For this purpose, the extracts were evaporated under a stream of nitrogen and lled up to 50 mL (arils) and 100 mL (pericarp) with methylene chloride. Single-use cartridges were lled with 2 g of polyamide and activated with 25 mL methanol and 50 mL methylene chloride. Aliquots of 5 mL (pericarp) and 10 mL (arils) were applied to the sorbent and after washing with 50 mL of methylene chloride, xanthones were eluted with 100 mL of methanol. After evaporation to dryness in vacuo at 30 C, the eluates were dissolved in 3 mL of methanol, membrane-ltered (0.45 lm) and used for LCMS analysis. For the isolation of xanthones from the functional beverage, the extraction was performed as previously described, using aliquots of 30 mL. After evaporation, the residues were dissolved in 3 mL of methanol, membrane-ltered (0.45 lm, Whatman, Dassel, Germany) and directly used for quantication with HPLCDAD. Prior to the identication of xanthones with LCMS, beverage extracts were made up to 50 mL and aliquots of 10 mL were puried with polyamide as described above. 2.3. HPLC analyses Separation of xanthones was performed using an Agilent HPLC series 1200 system (Agilent, Waldbronn, Germany) with ChemStation software, a model G1379 degasser, a model G1312B binary gradient pump, a model G1367D thermoautosampler, a model G1316B column oven and a model G1315C diode array detector. The column used was a 50 4,6 Zorbax Eclipse XDB from Agilent (Waldbronn, Germany) operated at a temperature of 25 C. The mobile phase consisted of 2% acetic acid in water (eluent A) and 0.5% acetic acid in acetonitrile (eluent B) using a gradient program as follows: 5060% B (20 min), 6070% B (35 min), 70 100% B (5 min), 100% B isocratic (3 min), 1000% B (2 min) at a ow rate of 0.6 mL/min. Total run time was 65 min. The injection volume was 8 lL for all samples. Xanthones were monitored at

2. Materials and methods 2.1. Chemicals, plant material and functional mangosteen product Reagents and solvents were purchased from VWR (Darmstadt, Germany) and were of analytical or HPLC grade. The a-mangostin standard was from ABCR Dr. Braunagel (Karlsruhe, Germany).

J. Wittenauer et al. / Food Chemistry 134 (2012) 445452

447

254 nm and UV/vis spectra were recorded in the range of 200 600 nm. Xanthones were identied by UV/vis and mass spectra and quantied using a calibration curve of the a-mangostin standard. The quantication of further, structurally related xanthones was performed applying a molecular weight correction factor (Chandra, Rana, & Li, 2001). All determinations were carried out in triplicate. 2.4. LCMS analyses LCMS analyses were performed with an Agilent HPLC series 1100 system equipped with ChemStation software, a model G1322A degasser, a model G1312A binary gradient pump, a model G1329/G1330A thermoautosampler, a model G1316A column oven and a model G1315A diode array detector. The column, eluents and HPLC parameters were the same as described above. The HPLC-system was coupled on-line with a Bruker (Bremen, Germany) model esquire 3000 + ion trap mass spectrometer tted with an ESI source. Data acquisition and processing were performed using Esquire Control Software. Negative ion mass spectra of the column eluate were recorded in the range of m/z 501000. Nitrogen was used both as drying gas at a ow rate of 10 L/min and as nebulising gas at a pressure of 60 psi. The nebuliser temperature was set at 365 C. Collision-induced dissociation spectra were obtained with a fragmentation amplitude of 1.2 V. Helium was used as the collision gas. 3. Results and discussion As prenylated xanthones are the major secondary plant metabolites in mangosteens, they are supposed to be responsible for the positive health effects of the fruits, thus being the basis for the claimed effectiveness of functional products from mangosteens. For most commercial mangosteen products including the analysed functional beverage, processing of mangosteens is already realised in their cultivation areas, commonly using the entire fully ripened fruits including the edible aril segments and the pericarp (Bin Osman & Milan, 2006; Kanchanapoom & Kanchanapoom, 1998; Morton, 1987). Since mangosteen pericarp has only been consumed in its country of origin so far and has not been used for human consumption to a signicant extent within the European Community before May 1997, it is still considered a novel food despite its long-lasting utilisation as a food and in traditional medicine. This implies that products containing mangosteen pericarp need to be authorised according to the Novel Food Directive No. 258/97. Due to its strict interpretation, thus creating economic trade barriers, revision of the legislation is currently under discussion (European Commission, 2011). According to an updated draft version, exotic fruits with a signicant history of use outside the EU will be permitted to undergo a simplied application procedure based on a safety assessment considering their history of safe use. Therefore, one objective of the present study was to provide evidence of essential equivalence of mangosteen pericarp composition with particular emphasis on its xanthone prole as compared to the edible aril segments. Besides the characterisation and quantication of these constituents in both plant parts, a functional beverage, produced from purees made from the whole mangosteen fruits, was also included in the xanthone proling. For this purpose, extracts of the mangosteen pericarp, the edible aril segments and the functional beverage were analysed with HPLCDAD and LCMS. Due to the limited availability of xanthone references and because UV/vis spectral data do not allow unambiguous identication of individual components, HPLC coupled to mass spectrometry proved to be an indispensable tool for peak assignment. Since limited fragmentation facilitated the tentative

identication of the separated xanthones (Zarena & Sankar, 2009), ESI techniques have been conrmed to be perfectly suitable for xanthone characterisation due to their soft ionisation yielding mainly integral molecular ions. The LCMS data of all identied xanthones are summarised in Table 1. 3.1. Identication of xanthones from pericarp, aril segments and the functional beverage with LCMSn Fig. 1 shows the HPLC proles of xanthones extracted from mangosteen pericarp, aril segments and the functional beverage. Polarity and hence, the retention time of the compounds was affected by the position of hydroxyl and prenyl groups varying the xanthen-9-one core. Similar ndings have already been reported in a previous study by Chaivisuthangkura et al. (2009) evaluating a method for the determination of xanthones in mangosteen fruits with HPLCDAD. In agreement with our study, 254 nm was chosen as detection wavelength for the ngerprint chromatogram and quantication of the xanthones. For ESI-MS analysis, xanthones were recorded in the negative ion mode. Contrary to the positive mode, negative ionisation resulted in increased fragmentation of the xanthones in the MS/MS spectra, thus facilitating their identication and structure elucidation. In agreement with Zhou, Han, Song, Qiao, and Xu (2008) as well as Da Costa et al. (2000) fragmentation pathways of the polyprenylated xanthones were found to follow general trends. All compounds showed abundant [MH] ions and yielded successive loss of prenyl residues C4H8 (56 Da) in the MS/MS spectra. 3.1.1. Identication of xanthones from pericarp and aril segments Astonishingly, the pericarp and aril segments of mangosteens exhibited similar ngerprints each consisting of two major and ve minor components. Based on molecular weights, order of elution, comparison with a-mangostin standard, and fragmentation, all 7 compounds belonging to the xanthones were tentatively identied. Concordant with literature data, the predominant peak could be assigned to a-mangostin (compound 6) (Chaivisuthangkura et al., 2009; Jung et al., 2006; Walker, 2007). Fig. 2 shows the mass spectrum and the suggested fragmentation pathway for a-mangostin. Fragmentation of m/z 409 for a-mangostin yielded a de-methylated product ion at m/z 394. In contrast to most other detected fragments, the product ion at m/z 394 exhibited an even mass. Since xanthones do not contain three bonding atoms such as nitrogen or phosphorus, the even mass leads to the assumption, that the dissociation of the CO-bond followed a homolytic mechanism. The resulting radical ion showed a relatively high intensity that may be due to resonance stabilisation. Similar fragmentation patterns with a homolytic cleavage and radical fragments of even masses were also observed for the other xanthones. The predominant peak in the MS spectrum of a-mangostin is at m/z 351, resulting from a following ring closure under loss of a C3H7 radical (43 Da). As a competing reaction, the de-methylated radical ion reacted under loss of a C4H7 radical (55 Da) from a prenyl group, yielding a product ion at m/z 339. Under the applied mass spectrometric conditions, the formation of a de-methoxylated product ion (32 Da) at m/z 377 was also observed. In addition, the loss of the second prenyl group in allylic position (56 Da) could be observed in the MS3-experiments. The fragmentation reactions of a-mangostin are comparable to those reported by Zhou et al. (2008), who analysed polyprenylated xanthones from Garcinia xipshuanbannaensis Y.H. Li. extracts by HPLCESI-QTOF-MSn. The second major peak of mangosteen pericarp (compound 2) was assigned to cmangostin. Compared to a-mangostin, the c-form is characterised by the presence of a hydroxyl group at position 7 instead of a methyl group. Therefore, the elimination of prenyl moieties in allylic position may be the preferred reaction. Mass spectrometric

448

J. Wittenauer et al. / Food Chemistry 134 (2012) 445452

Table 1 UV spectra and characteristic ions of xanthones extracted from mangosteen pericarp, aril segments and the analysed functional beverage. Compound Retention time (min) 15.8 Xanthone Structure HPLC DAD kmax (nm) 243, 263, 308, 377 [MH] m/z HPLCESI(-)-MSn experiment m/z (% base peak) MS2 [325]: 310 (100), 267 (20), 311 (13) MS3 [325 ? 310]: 267 (100), 255 (11)

1,7-Dihydroxy-3-methoxy2-(3-methylbut-2enyl)xanthon

325

19.0

c-Mangostin

243, 260, 317, 364

395

MS2 [395]: 283 (100), 339 (21), 326 (16), 297 (12), 351 (11), 271 (10), 395 (10)

21.1

8-Deoxygartanin

244, 259, 322, 374

379

MS2 [379]: 324 (100), 281 (91) MS3 [397 ? 324]: 281 (100), 269 (20)

24.0

1,3,7-Trihydroxy-2,8-di(3-methylbut-2enyl)xanthon

241, 365, 314, 378

379

MS2 [379]: 310 (100), 324 (44), 267 (35), 281 (29), 323 (19) MS3[379 ? 310]: 267 (100), 255 (15), 268 (11)

26.3

Gartanin

227, 258, 282, 349

395

MS2 [395]: 340 (100), 297 (78), 380 (31), 351 (28), 337 (13) MS3 [395 ? 340]: 297 (100), 285 (13)

28.3

a-Mangostin

247, 317, 354

409

MS2 [409]: 351 (100), 394 (75), 377 (43), 339 (21) MS3 [409 ? 351]: 295 (100), 307 (75), 282 (43), 308 (36), 296 (33)

35.2

Garcinon E

245, 260, 321, 362

463

MS2 [463]: 351 (100), 463 (30), 339 (22), 407 (21), 365 (18), 352 (12), 419 (12), 408 (11) MS3 [463 ? 351]: 296 (100)

analysis revealed a predominant peak at m/z 283 that may correspond to a loss of two C4H8 (56 Da) fragments originating from the prenyl groups. A loss of a single prenyl group in allylic position (56 Da) and a subsequent loss of the second prenyl group under formation of a heterocyclic ring (42 Da) and a fragment at m/z 297 could be also observed. Moreover, collision induced fragmentation of the [MH] ion yielded a fragment at m/z 326 caused by a loss of a prenyl radical C5H9 (69 Da). Similar to a-mangostin, the resulting radical ion is resonance-stabilised. A further fragment at m/z 271 resulted from the loss of both prenyl groups, one group in allylic and the other in benzylic (68 Da) position. The other components of the ngerprint of mangosteen pericarp and aril segments were present in lower concentrations. Fragmentation pattern of compound 1 was similar to a-mangostin with a de-methylated product ion at m/z 310 (15 Da) and a presumptive subsequent ring closure leading to m/z 267 (58 Da), which could be corroborated by the MS3 fragmentation experiments. Based on retention time, molecular weight and fragmentation pat-

tern, compound 1 was assigned to 1,7-dihydroxy-3-methoxy-2-(3methylbut-2-enyl)xanthone. Compound 3 showed an [MH] ion of m/z 379 and was tentatively assigned to 8-deoxygartanin. CID of the [MH] ion resulted in the loss of a C4H7 radical (55 Da), that can be attributed to an allylic loss of the prenyl group, yielding a prominent fragment at m/z 324. Unlike fragmentation reactions of other compounds, the loss of more than one prenyl group (112 Da, 124 Da) could not be observed. This might be attributed to the structure of 8deoxygartanin, with two prenyl groups at the same aromatic ring. Fragmentation of one prenyl group leads to a loss of aromaticity. Consequently, formation of stable fragments after further elimination of the second prenyl group is impossible. Compound 4 was detected exhibiting an [MH] ion of m/z 379 and tentatively identied as 1,3,7-trihydroxy-2,8-di(3-methylbut2-enyl)xanthone. As can be seen in the UV-chromatogram, peak 4 comprises two compounds, while only one single peak could be detected in the ion chromatogram, indicating the presence of

J. Wittenauer et al. / Food Chemistry 134 (2012) 445452

449

Pericarp
100
1 4 2 3 5

Aril Segments
100
6 4 1 2 3 5 7

mAU
0

Functional beverage
100
2 1 3 4 5

0 10

20

30

40

min
Fig. 1. Separation of xanthones extracted from mangosteen pericarp, aril segments and the functional beverage by high-performance liquid chromatography (254 nm). Peak assignment (1) 1,7-dihydroxy-3-methoxy-2-(3-methylbut-2-enyl)xanthon, (2) c-mangostin, (3) 8-deoxygartanin, (4) 1,3,7-trihydroxy-2,8-di(3-methylbut-2-enyl)xanthon, (5) gartanin, (6) a-mangostin, (7) garcinon E.

-H O O HO O m/z = 409 OH OH

-H O
- 15 Da

-H O
- 43 Da

OH O HO

OH

O HO O m/z = 394 OH

- CH3

- C3H7

O m/z = 351

OH

- CH3OH - 32 Da

- C4H7 - 55 Da
100

350,8

-H O OH

-H O O OH

+
80

393,8

Relative intensity (%)

60

O m/z = 377

OH

376,8
40

HO

O m/z 339

OH

20

339

0 300 320 340 360 380 400 420 440

m/z

Fig. 2. Mass spectra and postulated fragmentation pathway of a-mangostin. The proposed fragmentation pathway is based on literature data of similar xanthones. In case of unavailable fragmentation schemes, the fragment ion was identied by mass value only.

isomeric forms. Since 1,3,7-trihydroxy-2,8-di(3-methylbut-2enyl)xanthone is devoid of stereogenic centres, and the UV-spectra of both peak apexes are identical, this peculiarity needs deeper investigation. Fragmentation pattern of 1,3,7-trihydroxy-2,8-di(3methylbut-2-enyl)xanthon exhibits the same fragments as described above with the preferred fragmentation of one prenyl group. The main peak at m/z 379 corresponds to a loss of a prenyl moiety C5H9 (69 Da). Peak 5 showed an [MH] ion of m/z 395 that could be assigned to gartanin. The presence of gartanin in mangosteen pericarp has previously been described by several authors (Govindachari,

Kalyanaraman, Muthukumaraswamy, & Pai, 1971; Jung et al., 2006; Mahabusarakam & Wiriyachitra, 1987; Zarena & Sankar, 2009). In agreement with literature ndings, gartanin eluted prior to a-mangostin. For this reason, peak assignment was mainly conrmed by the elution order and molecular weight. Besides a typical loss of a C4H7 radical (55 Da) originating from a prenyl group, the most prominent product ion at m/z 297 (98 Da) resulting from the loss of a C3H7 radical (43 Da) under formation of a heterocylic ring could be observed. Compound 7 exhibiting an [MH] ion at m/z 463 was detected and tentatively identied as garcinon E. Since the structure of

450

J. Wittenauer et al. / Food Chemistry 134 (2012) 445452

garcinon E exhibits three prenyl groups, the molecule is more lipophilic than the other xanthones, thus displaying a longer retention. Moreover, the preferred fragmentation reaction of garcinon E is a loss of two fragments, each from different prenyl groups. The main peak at m/z 351 corresponds to a loss of two C4H8 (56 Da) fragments from the prenyl groups. Furthermore, fragmentation at the benzylic position and ring closure reactions led to fragments characterised by a loss of two fragments of prenylic origin (124 Da, 98 Da). Altogether, fragmentation of garcinon E turned out to be similar to c-mangostin. Although previous studies of mangosteen pericarp revealed the occurrence of isomangostin, 9-hydroxycalabaxanthon and mangostin (Mahabusarakam & Wiriyachitra, 1987; Zarena & Sankar, 2009), their unambiguous presence could not be conrmed. 3.1.2. Identication of xanthones from the functional beverage Analysis of the extract from the functional beverage by LCMSn showed good agreement with the xanthone pattern of the pericarp. a-Mangostin revealed to be the predominant xanthone, followed by c-mangostin. Other characteristic xanthones were only detected at low concentrations. 3.2. Quantication of the major xanthones in pericarp, aril segments and the functional beverage Only few studies dealt with the quantication of xanthones in mangosteens and, in most of these studies, the origin and the analysed fruit part has not precisely been assigned to a specic plant part. Only one single study by Zarena and Sankar (2009) investigated the composition of a mangosteen pericarp extract. Since supercritical carbon dioxide extraction was applied, comparison with data obtained in the present work is difcult. Table 2 lists the determined amounts of the identied xanthones in pericarp, aril segments and the functional beverage as well as the total xanthone contents. The total xanthone content, as calculated by summarising individual amounts of all constituents, was 1700.26 40.47 mg/100 g on fresh weight (FW), 106.87 3.15 mg/100 g FW and 18.79 0.49 mg/100 mL, for the mangosteen pericarp, the aril segments and the functional beverage, respectively. Despite the large diversity of xanthones that has been found in the aril segments, xanthone contents are 16 times lower than in the pericarp. Compared to the fruit parts, the analysed functional beverage contained even lower concentrations of xanthones, which is attributable to the low dosage of mangosteen puree (<5%) in the recipe of the nal product. According to the producers information, mangosteen puree is produced from the whole fully ripe fruits after blanching and milling to obtain a smooth pulp including the juicy non-lignied red-coloured pericarp. The fruit puree is nally blended with water and eight further fruit purees and juices, respectively. In addition to total xanthone contents in mangosteen pericarp, aril segments and the functional beverage, xanthone patterns of the different fruit parts displayed slight differences. While a-

mangostin represented the major xanthone in both parts of the fruit and in the functional beverage, the second major xanthone in mangosteen pericarp was c-mangostin, followed by the minor components gartanin, 8-deoxygartanin, garcinon E, 1,7-dihydroxy-3-methoxy-2-(3-methylbut-2-enyl)xanthone and 1,3,7-trihydroxy-2,8-di(3-methylbut-2-enyl)-xanthone. In contrast, aril segments from mangosteen showed a deviating concentration order of the identied minor xanthones. Besides 1,3,7-trihydroxy-2,8-di(3-methylbut-2-enyl)-xanthone as the second major xanthone, concentration of minor xanthones decreased in the order 1,7-dihydroxy-3-methoxy-2-(3-methylbut-2-enyl)xanthone, garcinon E, c-mangostin, gartanin and 8-deoxygartanin. In the pericarp, all identied xanthones were present at higher amounts than in the aril segment, except 1,3,7-trihydroxy-2,8-di(3-methylbut-2-enyl)xanthone. Concentration order of the different xanthones in the functional beverage turned out to be similar to that of the pericarp. To estimate the intake of xanthones with the usual consumption of one mangosteen fruit and by the ingestion of the functional beverage, respectively, comparison of major xanthone contents was conducive. For this purpose, contents of a-mangostin, cmangostin and 1,3,7-trihydroxy-2,8-di(3-methylbut-2-enyl)xanthone in arils, pericarp and the beverage were determined. Due to their very low concentrations minor xanthones have not been considered. Quantication of the most prominent xanthones in extracts from mangosteen aril segments resulted in an a-mangostin content of 40.59 1.62 mg/100 g FW and a c-mangostin content of 8.25 0.24 mg/100 g FW. Amounts of 1,3,7-trihydroxy-2,8-di(3methylbut-2-enyl)xanthone in the aril segments were 25.76 0.68 mg/100 g FW. Compared to the pericarp, the a- and c-mangostin contents in the aril segments were 37 and 29 times lower, respectively, whereas the concentration of 1,3,7-trihydroxy-2,8-di(3-methylbut-2-enyl)xanthone in mangosteen arils was in the range of the pericarp. Considering the consumption of the aril segments of one single Thai mangosteen (30 g), it results in an intake of 12.2 mg, 7.7 mg and 2.5 mg of a-mangostin, 1,3,7-trihydroxy-2,8-di(3-methylbut2-enyl)-xanthone and c-mangostin, respectively. Compared with the recommended daily dose of the functional beverage (90 mL), the content of the predominant a-mangostin in the functional beverage is similar to that of aril segments of one single Thai mangosteen. Comparable amounts were also determined for 1,3,7-trihydroxy-2,8-di(3-methylbut-2-enyl)xanthone and c-mangostin. Consequently, the ingestion of 90 mL of the functional beverage corresponds to the consumption of carefully peeled edible aril segments of one single mangosteen. Moreover, 90 mL of the beverage contain the same amounts of the major xanthones a- and cmangostin as 0.9 g of pericarp. Accordingly, only minor amounts of pericarp ingested with the peeled aril segments are needed to attain an intake in the range of the recommended daily dose of the beverage. As a consequence of these ndings, the bioavailability of xanthones from fruits and functional drinks should be an

Table 2 Contents of xanthones in mangosteen pericarp, aril segments and the analysed functional beverage. Xanthone Pericarp mg/100 g DM 1,7-Dihydroxy-3-methoxy-2-(3-methylbut-2-enyl)xanthon c-Mangostin 8-Deoxygartanin 1,3,7-Trihydroxy-2,8-di-(3-methylbut-2-enyl)xanthon Gartanin a-Mangostin Garcinon E Total amount 99.92 2.41 860.17 17.46 141.90 2.26 54.17 1.58 199.46 3.50 3323.88 82.18 137.09 5.27 4816.59 114.65 mg/100 g FW 35.27 0.85 303.64 6.16 50.09 0.80 19.12 0.56 70.41 1.24 1173.33 29.01 48.39 1.86 1700.26 40.47 Aril segments mg/100 g DM 65.88 1.28 43.39 1.27 26.39 0.45 135.57 3.56 28.92 0.58 213.63 8.53 48.70 0.88 562.48 16.56 mg/100 g FW 12.52 0.24 8.25 0.24 5.01 0.09 25.76 0.68 5.49 0.11 40.59 1.62 9.25 0.17 106.87 3.15 Functional beverage mg/100 mL 0.48 0.01 3.01 0.06 0.67 0.01 0.53 0.01 0.85 0.01 12.35 0.39 0.83 0.00 18.79 0.49

J. Wittenauer et al. / Food Chemistry 134 (2012) 445452

451

issue of further studies to determine the efcacy of xanthones ingested from different processed food products. 4. Summary and conclusions To the best of our knowledge, this investigation undoubtedly demonstrated for the rst time that mangosteen aril segments contain a complex mixture of xanthone species. The xanthone patterns of mangosteen pericarp as well as the aril and the functional beverage, are in good agreement, although differing in their quantitative composition. In contrast to mangosteen pericarp, the aril segments and the functional beverage contain considerably lower concentrations of individual xanthones. For the consumption of mangosteen fruits, the aril segments are manually separated from the pericarp. Thereby, mangosteen pericarp adhering to the aril segments are ingested. As a consequence, it is assumed that mangosteen pericarp was already consumed in noteworthy amounts prior to the legal validity of the Novel food regulation. Therefore, the extremely fastidious separation of pericarp and aril segments was a prerequisite for an accurate quantication of individual compounds in the isolated aril segments. However, taking into consideration the recommended daily dose of 90 mL of the functional beverage investigated in this study, the intake of pericarp constituents with the functional beverage corresponds to the consumption of less than 1 g of pericarp. Even the consumption of pure aril segments of one single mangosteen (30 g), sensu stricto constituting the edible part of the fruit, equals the daily recommended dose of the beverage. Moreover, numerous studies dealing with acute and subchronic toxicity of mangosteen pericarp extract, whole fruit, seed and seed oil, respectively, did not provide evidence of toxic risks (Ajayi, Oderinde, Ogunkoya, Egunyomi, & Taiwo, 2007; Hutadilok-Towatana, Reanmongkol, Wattanapiromsakul, & Bunkrongcheap, 2010; Settheetham & Ishida, 1995). Accordingly, food supplements based on the entire mangosteen fruit and with standardised a-mangostin contents enjoy a long-standing popularity in the USA. Therefore, and due to the traditional consumption of integral mangosteen fruits in their countries of origin, a history of safe use may be concluded. Remarkably, despite their xanthone C-glucoside contents being indicative of processing peel and the entire fruit into purees and concentrates derived from Mangifera indica L., relevant consumption of xanthones from the non-edible peels with mango juice, nectar and puree is a well established practice obviously complying with the Council Directive 2001/112/EC without being considered as novel foods (Berardini et al., 2005; Schieber, Ullrich, & Carle, 2000). Consequently, the novel food status of whole mangosteen products is inconsistent with the categorisation of mango products processed from unpeeled fruits. Besides food applications, the bioactive compounds of mangosteen also bear a large potential for pharmaceutical and cosmetic products. In particular, the antioxidative and antimicrobial properties of prenylated and oxygenated xanthones appear to be suitable for cosmetic formulations such as anti-aging emulsion or liposome preparations. The high potential of mangosteen extracts has already been recognised by the pharmaceutical and cosmetic industry as demonstrated by recently launched products such as a mouthwashes or different herbal cosmetics. Supported by the scientic evidence of the pharmaceutical and probably anti-aging activities, the interest in mangosteen extracts will be further increased in future. References
Ajayi, I. A., Oderinde, R. A., Ogunkoya, B. O., Egunyomi, A., & Taiwo, V. O. (2007). Chemical analysis and preliminary toxicological evaluation of Garcinia mangostana seeds and seed oil. Food Chemistry, 101, 9991004.

Berardini, N., Fezer, R., Conrad, J., Beifuss, U., Carle, R., & Schieber, A. (2005). Screening of mango (Mangifera indica L.) cultivars for their contents of avonol O- and xanthone C-glycosides, anthocyanins, and pectin. Journal of Agricultural and Food Chemistry, 53, 15631570. Bunsiri, A., Ketsa, S., & Paull, R. E. (2003). Phenolic metabolism and lignin synthesis in damaged pericarp of mangosteen fruit after impact. Postharvest Biology and Technology, 29, 6171. Bennett, G. J., & Lee, H. H. (1989). Xanthones from Guttiferae. Phytochemistry, 28, 967998. Bin Osman, M., & Milan, A. R. (2006). Mangosteen Garcinia mangostana. Southampton: Southampton Centre for Underutilised Crops, University of Southampton. Chaivisuthangkura, A., Malaikaew, Y., Chaovanalikit, A., Jaratrungtawee, A., Panseeta, P., Ratananukul, P., et al. (2009). Prenylated xanthone composition of Garcinia mangostana (mangosteen) fruit hull. Chromatographia, 69, 315318. Chandra, A., Rana, J., & Li, Y. (2001). Separation, identication, quantication and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLCMS. Journal of Agricultural and Food Chemistry, 49, 35153521. Da Costa, C. T., Dalluge, J. J., Welch, M. J., Coxon, B., Margolis, S. A., & Horton, D. (2000). Characterization of prenylated xanthones and avanones by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. Journal of Mass Spectrometry, 35, 540549. Dangcham, S., Bowen, J., Ferguson, I. B., & Ketsa, S. (2008). Effect of temperature and low oxygen on pericarp hardening of mangosteen fruit stored at low temperature. Postharvest Biology and Technology, 50, 3744. European Commission (2011). Novel Food Review of Regulation (EC) 258/97. Available from: http://ec.europa.eu/food/food/biotechnology/novelfood/ initiatives_en.htm [last update 29.07.11]. Ee, G. C., Daud, S., Tauq-Yap, Y. H., Ismail, N. H., & Rahmani, M. (2006). Xanthones from Garcinia mangostana (Guttiferae). Natural Product Research, 20, 10671073. Govindachari, T. R., Kalyanaraman, P. S., Muthukumaraswamy, N., & Pai, B. R. (1971). Xanthones of Garcinia mangostana Linn. Tetrahedron, 27, 39193926. Hutadilok-Towatana, N., Reanmongkol, W., Wattanapiromsakul, C., & Bunkrongcheap, R. (2010). Acute and subchronic toxicity evaluation of the hydroethanolic extract of mangosteen pericarp. Journal of Medicinal Plants Research, 4, 969974. Ji, X., Avula, B., & Khan, I. A. (2007). Quantitative and qualitative determination of six xanthones in Garcinia mangostana L. by LCPDA and LCESI-MS. Journal of Pharmaceutical and Biomedical Analysis, 43, 12701276. Jung, H.-A., Su, B.-N., Keller, W. J., Mehta, R. G., & Kinghorn, A. D. (2006). Antioxidant xanthones from the pericarp of Garcinia mangostana (mangosteen). Journal of Agricultural and Food Chemistry, 54, 207720852. Kanchanapoom, K., & Kanchanapoom, M. (1998). Mangosteen. In P. E. Shaw, H. T. Chan, & S. Nagy (Eds.), Tropical and subtropical fruits (pp. 191216). Aubundale: Agscience. Ketsa, S., & Atantee, S. (1998). Phenolics, lignin, peroxidase activity and increased rmness of damaged pericarp of mangosteen fruit after impact. Postharvest Biology and Technology, 14, 117124. Ketsa, S., & Koolpluksee, M. (1993). Some physical and biochemical characteristics of damaged pericarp of mangosteen fruit after impact. Postharvest Biology and Technology, 2, 209215. Mahabusarakam, W., & Wiriyachitra, P. (1987). Chemical constituents of Garcinia mangostana. Journal of Natural Products, 50, 474478. Morton, J. (1987). Mangosteen. In J. F. Morton (Ed.), Fruits of warm climates (pp. 301304). Florida: Florida Flair Books. Nilar, L. J. H. (2002). Xanthones from the heartwood of Garcinia mangostana. Phytochemistry, 60, 541548. Obolskiy, D., Pischel, I., Siriwatanametanon, N., & Heinrich, M. (2009). Garcinia mangostana L.: A phytochemical and pharmacological review. Phytotherapy Research, 23, 10471065. Palapol, Y., Ketsa, S., Stevenson, D., Cooney, J. M., Allan, A. C., & Ferguson, I. B. (2009). Colour development and quality of mangosteen (Garcinia mangostana L.) fruit during ripening and after harvest. Postharvest Biology and Technology, 51, 349353. Pedraza-Chaverri, J., Crdenas-Rodrguez, N., Orozoco-Ibarra, M., & Prez-Rojas, J. M. (2008). Medicinal properties of mangosteen (Garcinia mangostana). Food and Chemical Toxicology, 46, 32273239. Peres, V., Nagem, T. J., & de Oliveira, F. F. (2000). Tetraoxygenated naturally occurring xanthones. Phytochemistry, 55, 683710. Schieber, A., Berardini, N., & Carle, R. (2003). Identication of avonol and xanthone glycosides from mango (Mangifera indica L. Cv. Tommy Atkins) peels by highperformance liquid chromatographyelectrospray ionization mass spectrometry. Journal of Agricultural and Food Chemistry, 51, 50065011. 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 Science and Emerging Technologies, 1, 161166. Settheetham, W., & Ishida, T. (1995). Study of genotoxic effects of antidiarrheal medicinal herbs on human cells in vitro. Asian Journal of Tropical Medicine and Public Health, 26, 306310. Suksamrarn, S., Suwannapoch, N., Phakhodee, W., Thanuhiranlert, J., Ratananukul, P., Chimnoi, N., et al. (2003). Antimycobacterial activity of prenylated xanthones from the fruits of Garcinia mangostana. Chemical and Pharmaceutical Bulletin, 51, 857859. Walker, E. B. (2007). HPLC analysis of selected xanthones in mangosteen fruit. Journal of Separation Science, 30, 12291234.

452

J. Wittenauer et al. / Food Chemistry 134 (2012) 445452 Characterization by HPLC/LCESI-MS. The Journal of Supercritical Fluids, 49, 330337. Zhou, Y., Han, Q.-B., Song, J.-Z., Qiao, C.-F., & Xu, H.-X. (2008). Characterization of polyprenylated xanthones in Garcinia xipshuanbannaensis using liquid chromatography coupled with electrospray ionization quadrupole time-ofight tandem mass spectrometry. Journal of Chromatography A, 1206, 131139.

Weecharangsan, W., Opanasopit, P., Sukma, M., Ngawhirunpat, T., Sotanaphun, U., & Siripong, P. (2006). Antioxidative and neuroprotective activities of extracts from the fruit hull of mangosteen (Garcinia mangostana Linn.). Medical Principles and Practice, 15, 281287. Zarena, A. S., & Sankar, K. U. (2009). Supercritical carbon dioxide extraction of xanthones with antioxidant activity from Garcinia mangostana: