Phytochemical composition and antioxidant capacity of various botanical parts of the fruits of Prunus × domestica L. from the Lorraine region of Europe

Phytochemical composition and antioxidant capacity of various botanical parts of the fruits of Prunus × domestica L. from the Lorraine region of Europe

Food Chemistry 133 (2012) 697–706 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodch...

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Food Chemistry 133 (2012) 697–706

Contents lists available at SciVerse ScienceDirect

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

Phytochemical composition and antioxidant capacity of various botanical parts of the fruits of Prunus  domestica L. from the Lorraine region of Europe Farid Khallouki a,c, Roswitha Haubner a, Gerhard Erben b, Cornelia M. Ulrich a, Robert W. Owen a,⇑ a Division of Preventive Oncology, National Center for Tumor Diseases, Im Neuenheimer Feld 460/German Cancer Research Center(DKFZ), Im Neuenheimer Feld 581, 69120 Heidelberg, Germany b Core Facility, Molecular Structure Analysis, German Cancer Research Center(DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany c Natural Substances Biochemistry Laboratory, LBSN, FSTE, BP 509 Boutalamine, Errachidia, Morocco

a r t i c l e

i n f o

Article history: Received 22 August 2011 Received in revised form 6 December 2011 Accepted 23 January 2012 Available online 9 February 2012 Keywords: Prunus Rosaceae Mirabelle Chemoprevention Phenolic compounds Antioxidant capacity

a b s t r a c t Mirabelle plums represent a famous fruit from the Lorraine region, however little is known about their phytochemical composition. The oil of the fruit contained predominantly oleic acid (59%) and linoleic acid (29%). The total content of phenolic antioxidants in the whole fruits was 5.338 g/kg with 456 mg/ kg (9%), 701 mg/kg (13%) and 4159 mg/kg (78%) detected in the peels, flesh and pits respectively. The peels contained solely 3,4-dihydroxybenzoic acid (270 mg/kg) and rutin (186 mg/kg), the flesh exclusively echinoids (723 mg/kg), whereas the pits contained a rich variety of phenolic compounds (4.2 g/ kg) dominated by amygdalin (3.8 g/kg), but with significant contributions from vanillin (102 mg/kg), guajacyl-glycerin-coniferyl aldehyde isomers (87 mg/kg), dehydro-diconiferyl aldehyde (52 mg/kg), and vanillin diglucoside (48 mg/kg). Of the major phenolic compounds tested across a range of in vitro assays, rutin was the superior antioxidant. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Prunus  domestica (Rosaceae) is known as Myrobolan, Cherry plum or Mirabelle plum. This is the famous fruit of the Lorraine gastronomy region (north-east France), Saarland (Germany) and Luxembourg. Indeed this area has the strongest density of Mirabelle plum trees in the World and contributes about 80% of global production. The Mirabelle plum tree is, in the Lorraine region, what the olive-tree represents in the Mediterranean basin (Owen, Giacosa, et al., 2000) and the argan tree in Morocco (Khallouki et al., 2003). Mirabelle plums of the Lorraine region usually reach maturity between the 10th and 15th August. It is a plant which historically originates from the Caucasus region in West Asia and is one of the species of plum which is a French speciality and is closely allied to the damson and bullace. It is usually classified as a variety of Prunus instititia, although some regard it as a hybrid of Prunus cerasifera and Prunus domestica. The variety known as Mirabelle de Nancy is believed to have arrived in France from the East in the 15th century, where it is still highly rated. The Mirabelle of Metz (France) was first recorded in 1675, as sourced from the website http:// www.innvista.com/health/foods/fruits/plums.htm.

⇑ Corresponding author. Tel.: +49 6221 42 3317; fax: +49 6221 42 3375. E-mail address: [email protected] (R.W. Owen). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.01.071

In general, plum refers to the fruits of the genus Prunus, such as P. domestica, P. salicina, P. subcordiata, and P. insititia. Among P. domestica, the so-called prune is the dried fruit of some cultivars of P. domestica and therefore, these cultivars are referred to as ‘‘prune-making plum’’. Ethnobotanic data indicate that prunes are well-known as a healthy food (Stacewicz-Sapuntzakis, Bowen, Hussain, Damayanti-Wood, & Farnsworth, 2001) and have been used in India, in combination with other drugs, for the treatment of leucorrhoea, irregular menstruation and debility following miscarriage (Chopra, Nayar, & Chopra, 1956). Prunus kernels have been utilised in medicine for centuries; indeed, Avicenna controversially recommended the use of apricot bitter almond (Prunus amygdalus) oil in the treatment of tumours of the spleen, uterus, stomach and liver. Neochlorogenic acid (3-O-caffeoylquinic acid, 3-CQA) is reported in the literature as the major hydroxycinnamate in the plum fruits, followed by chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA), with low contributions from cryptochlorogenic acid (4-Ocaffeoylquinic acid, 4-CQA) (Herrmann, 1989; Nakatani et al., 2000; Slimestad, Vangdal, & Brede, 2009). The ratio obtained by HPLC as reported by Nakatani et al. (2000) was 78:18:4, respectively, for 3-CQA, 5-CQA and 4-CQA. In other reports, Raynal, Moutounet, and Souquet (1989) showed that 3-CQA was about half the amount of the total phenolics in the exocarp and pulp of fresh prunes (P. domestica).

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This is attributed to the content of caffeoyl quinic acid isomers (CQAI), but several studies indicate a contribution from as yet unidentified antioxidants in Prunus extracts (Kayano et al., 2003). CQAI have been described to prevent oxidation of human LDL (Rice-Evans, Miller, & Paganga, 1996), lower low-density lipoprotein (LDL) cholesterol in rat livers (Tinker, Davis, & Schneeman, 1994), show suppressive effects against chemical mutagens (Edenharder, Kurz, John, Burgard, & Seeger, 1994), scavenge both reactive oxygen and nitrogen species (Kono et al., 1997), induce formation of bone in postmenopausal women (Arjmandi et al., 2002) and possess antiproliferative and proapoptotic capacities (Fujii, Ikami, Xu, & Ikeda, 2006). A more recent report indicates the effect of an extract of Prunus salicina Lindl. cv. Soldam fruit on the viability and induction of apoptosis in MDA-MB-231 cells. As a result, an acetone extract of immature plums showed induction of apoptosis in MDA-MB-231 cells (Yu et al., 2007). In addition, in vitro binding of bile acids by peaches (Prunus persica) among other fruits, was determined using

Interesting compounds such as abscisic acid and lignan derivatives (Kikuzaki et al., 2004), procyanidins (Nunes et al., 2008), anthocyanidins namely cyanidin 3-rutinoside and peonidin 3-rutinoside (Usenika, Štampar, & Vebericˇ, 2009), along with C-alkyl isoflavones (Kosar et al., 2010) are among other major metabolites recently identified in P. domestica. With regard to phytochemical constituents from other parts of P. domestica L. Kaempferol and quercetin glycosides were also reported in the leaves and fruits of P. domestica L. and Prunuus salicina Lindley, but the total contents of these metabolites in the fruits were quite low at 20–52 mg/kg (Henning & Herrmann, 1980). Fruits and vegetables are known to contribute a large range of phenolic components to the diet, namely flavonoids, lignans, flavonolignans, tannins, coumarins, curcuminoids, echinoids, phenolic acids, and their derivatives (Kayano et al., 2003), and they show strong antioxidant activity (Cao, Booth, Sadowski, & Prior, 1998). In this context the antioxidant activity of prunes is very high in comparison to the antioxidant activities of other fruits and vegetables.

Abundance

4 2.5e+07 2.4e+07 2.3e+07 2.2e+07 2.1e+07 2e+07 1.9e+07 1.8e+07 1.7e+07 1.6e+07 1.5e+07 1.4e+07 1.3e+07 1.2e+07 1.1e+07 1e+07 9000000 8000000 7000000 6000000 5000000 4000000 3000000 2000000 1000000

3

2 5

1 10.00

15.00

20.00

25.00

30.00

35.00

Time (minutes) Fig. 1a. (a) GC–MS chromatogram of the oil extracted from the flesh of Prunus  domestica L. (Mirabelle) is shown. 1, palmitoleic acid; 2, palmitic acid; 3, linoleic acid; 4, oleic acid; 5, stearic acid.

1

6500000 6000000 5500000 5000000

Abundance

4500000 4000000 3500000 3000000

3

2500000 2000000 1500000

2

1000000 500000 38.00

40.00

42.00

44.00

46.00

48.00

50.00

52.00

54.00

56.00

58.00

Time (minutes) Fig. 1b. GC–MS chromatogram of the oil extracted from the flesh of Prunus  domestica L. (Mirabelle) is shown. 1, diacylglycerols; 2, c-tocopherol, 3, b-sitosterol.

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F. Khallouki et al. / Food Chemistry 133 (2012) 697–706 Table 1 Nano ESI-MS-MS of the phenolic compounds detected in the various botanical parts of the fruits of Prunus domestica L.

*

No*

Phenolic compound

[MH]

Nano ESI-MS-MS fragmentation (neg. ion m/z) Fragment ions

I II III IV

Benzoic acid (M = 122) p-Hydroxybenzaldehyde (M = 122) p-Hydroxybenzoic acid (M = 138) Vanillin (M = 152)

120.9 121.1 136.9 151.0

V

3,4-Dihydroxybenzoic acid (M = 154)

152.9

76.9 = [MH]–CO2 92.1 = [MH]–CHO 92.9 = [MH]–CO2 136.0 = [MH]–CH3 108.0 = [MH]–CH3 + CO 92.0 = [MH]–CH3 + CO + OH 108.8 = [MH]–CO2

VI

Vanillic acid (M = 168)

166.9

VII VIII

Gallic acid (M = 170) Syringaldehyde (M = 182)

169.1 180.8

IX

Syringic acid (M = 198)

196.9

X

Coniferyl aldehyde (M = 178)

177.1

XI

Dimethoxycinnamaldehyde (M = 208)

207.1

XII

Vanillin diglucoside (M = 476)

475.0

XIII XIV

p-Coumaroyl quinic acid (M = 338) Neochlorogenic acid (M = 354)

337.1 353.1

XV

Cryptochlorogenic acid (M = 354)

353.1

XVI

Chlorogenic acid (M = 354)

353.1

XVII

Rutin (M = 610)

609.1

XVIII

Amygdalin (M = 457)

456.2

XIX XX XXI

Dehydro-diconiferyl aldehyde (M = 356) Guajacyl-glycerin-coniferyl aldehyde (M = 374) Guajacyl-glycerin-coniferyl aldehyde (M = 374)

355.1 373.1 373.1

152.1 = [MH]–CH3122.9 = [MH]–CO2 108.0 = [MH]–CH3 + CO2 91.0 = [MH]–CH3 + CO2 + OH 125.1 = [MH]–CO2 165.7 = MH]–CH3 150.9 = [MH]–2CH3 122.8 = [MH]–2CH3 + CO 181.8 = [MH]–CH3166.8 = [MH]–2CH3 153.0 = [MH]–CO2 138.0 = [MH]–CH3 + CO2 122.9 = [MH]–2CH3 + CO2 120.9 = [MH]–CH3 + CO2 + H2O 106.1 = [MH]–2CH3 + CO2 + H2O 162.0 = [MH]–CH3 134.0 = [MH]–CH3 + CO 192.1 = [MH]–CH3 177.0 = [MH]–2CH3 149.0 = [MH]–2CH3 + CO 313.1 [MH]–glucose 151.1 [MH]–2glucose 163.1 = [MH]–quinic acid 191.1 = [MH]–caffeic acid 179.1 = [MH]–quinic acid 191.1 = [MH]–caffeic acid 179.1 = [MH]–quinic acid 191.1 = [MH]–caffeic acid 179.1 = [MH]–quinic acid 463.1 = [MH]–rhamnose 301.1 = [MH]–rhamnose + glucose 294.1 = [MH]–glucose 133.1 = [MH]–2glucose See Fig. 6 See Fig. 7 See Fig. 7

Identifier in Fig. 2.

Table 2 GC–EI-MS data for the TMS-ether derivatives of non-conjugated phenolic compounds detected in the various botanical parts of the fruits of Prunus domestica L. *

No

I II III IV V VI VII VIII IX X XI XIX XX XXI *

Phenolic compound

Benzoic acid p-Hydroxybenzaldehyde p-Hydroxybenzoic acid Vanillin 3,4-Dihydroxybenzoic acid Vanillic acid Gallic acid Syringaldehyde Syringic acid Coniferyl aldehyde 3,5-Dimethoxycinnamaldehyde Dehydro-diconiferyl aldehyde Guajacyl-glycerin-coniferyl aldehyde Guajacyl-glycerin-coniferyl aldehyde

TMS groupsa

1 1 2 1 3 2 4 1 2 1 1 2 3 3

M+ (calc.)

194 194 282 224 370 312 458 254 342 250 280 500 590 590

m/z (rel. abundance%) M+

Fragment ionsa

194(9) 194(77) 282(26) 224(33) 370(68) 312(68) 458(97) 254(32) 342(80) 250(94) 280(91) 500(26) 590(0.8) 590(0.7)

179(100), 135(43), 105(55) 179(100), 151(62) 267(100), 223(64), 193(48) 209(48), 194(100) 355(36), 311(22), 281(12), 267(6), 223(9), 193(100) 297(100), 282(29), 267(65), 253(41), 223(43) 443(33), 281(100), 179(10) 239(46), 224(100) 327(100), 312(70), 297(54), 283(19), 253(33), 223(19) 235(38), 220(100), 192(51) 265(36), 250(55) 222(100), 179(19) 485(4), 470(25), 455(6), 440(5), 410(100), 397(13), 380(22), 209(9), 103(27) 366(1.1), 323(2.9), 297(100), 209(4.9), 103(3.7) 366(0.8), 323(3.1), 297(100), 209(5.6), 103(3.5)

Identifier in Fig. 2. Base peak in bold face.

a

a mixture of bile acids secreted in human bile at a duodenal physiological pH of 6.3. The bile acid binding of peaches were signifi-

cantly higher than for grapes, pears and apricots, and maybe related to its phytonutrients, and antioxidant polyphenols. These

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F. Khallouki et al. / Food Chemistry 133 (2012) 697–706 CHO

COOH II

I

CHO

COOH

OCH3

OH

OH

IX OCH3 CH3O

CH3O

OH

CHO H

H

XII

H

OH

OH

XI OCH3 CH3O

OCH3

OH

HO

OH

O HO

O

C O

OH H

O

OH

H

H

OH

O

H

HO

O

O

C

OH

C O

H

H

H

H OH

OH OH

COOH OH XVI

HO

OH

O

C

OH

XV

XIV

XIII

O

COOH OH

COOH OH

COOH OH

OCH3

O

O

HO

OH

HO

OCH3 OH

OH

CHO

CHO

X

COOH

VIII

HO

OH

H

OH

CHO

VII OCH3

OH

OH

OH

COOH

VI

V

IV

III

COOH

COOH

OH

OH

OH

OH

OH HO

OH HO

HO

O HO

XVII

C OH

HO HO HO HO

OH

O

O

O

OH

O O O

HO

O

H 3C HO

O HO

XVIII

O CN

OH

H

HOH2C

O

XIX HO

C

CHO C H

OCH3

OCH3

H HOH2C

HO OCH3

C

CHO

H

H

HOH2C

O OH

XX

C

OCH3

C

CHO H

O OH

XXI HO

C

OCH3

OCH3

Fig. 2. 1, Benzoic acid; 2, p-hydroxybenzaldehyde; 3, p-hydroxybenzoic acid; 4, vanillin; 5, 3,4-dihydroxybenzoic acid; 6, vanillic acid; 7, gallic acid; 8, syringaldehyde; 9, syringic acid; 10, coniferyl aldehyde; 11, 3,5-dimethoxy-4-cinnamaldehyde; 12, vanillin diglucoside; 13, p-coumaroyl quinic acid; 14, neochlorogenic acid; 15, cryptochlorogenic acid; 16, chlorogenic acid; 17, rutin; 18, amygdalin; 19, dehydro-diconiferyl aldehyde; 20, guajacyl-glycerin-coniferyl aldehyde-1; 21, guajacyl-glycerinconiferyl aldehyde-2.

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UV 278 nm

250 200

Detector response

150 1

100

2

50 0

0

5

10

UV 340 nm

200

15

20

25

30

35

2

150 100 50 0 0

5

10

15

20

25

30

35

Time (minutes) Fig. 3. Analytical reverse-phase HPLC chromatogram of a methanol extract of the peels of Prunus  domestica L. (Mirabelle). 1, 3,4-dihydroxybenzoic acid; 2, rutin.

400

2

UV 278 nm

Detector response

300 200 100

1

0

0

800 UV 700 600 500 400 300 200 100 0 0

5

10

340 nm

3

5

15 2

20

3 5

4

10

4

15

25

30

35

5 20

25

30

35

Time (minutes) Fig. 4. Analytical reverse-phase HPLC chromatogram of a methanol extract of the flesh of Prunus  domestica L. (Mirabelle). 1, cis-neochlorogenic acid; 2, neochlorogenic acid; 3, p-coumaroyl quinate; 4, chlorogenic acid; 5, cryptochlorogenic acid.

Detector response

100 80 60 40 20 0

5

UV 278 nm

1 0

5

2

4

10

15

20

UV 340 nm

40 30 20 10 0 0

5

UV 250 nm

10

15

1

3

20

4

5

10

25

9 67 8

5

15

30

35

40

35

40

35

40

10

8

2

0

9

6 5

25 20 15 10 5 0

25

7

20

25

30

10

30

Time (minutes) Fig. 5. Analytical reverse-phase HPLC chromatogram of a methanol extract of the pits of Prunus  domestica L. (Mirabelle). 1, 3,4-dihydroxybenzoic acid; 2, vanillin diglucoside; 3, amygdalin; 4, vanillic acid; 5, vanillin; 6, guajacyl-glycerin-coniferyl aldehyde isomer-1; 7, guajacyl-glycerin-coniferyl aldehyde isomer-2; 8, coniferyl aldehyde; 9, 3,5-dimethoxycinnamaldehyde; 10, dehydro-diconiferyl aldehyde.

results point to the relative health promoting potential of prunus in chronic disease amelioration and more particularly in cancer chemoprevention (Kahlon & Smith, 2007).

In our ongoing research on the phytochemical constituents of various plant species, studies have been conducted on Prunus  domestica L. (Mirabelle). In this article, we report on the lipid

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Table 3a Amounts of phenolic compounds in the peel extract of Prunus domestica L. as determined by reverse-phase analytical HPLC–ESI-MS. Identifier in Fig. 2

Compound

Amount (mg/kg)

V XVII Total (mg/kg)

3,4-Dihydroxybenzoic acid Rutin

270 186 456

Table 3b Amounts of phenolic compounds in the flesh extract of Prunus domestica L. as determined by reverse-phase analytical HPLC–ESI-MS. Identifier in Fig. 2

Compound

Amount (mg/ kg)

XIII XIV

p-Coumaroyl quinic acid Neochlorogenic acid + cisneochlorogenic acid Cryptochlorogenic acid Chlorogenic acid

32 575

XV XVI Total (mg/kg)

22 94 723

Fe2Cl36H2O, hexane, methanol, N,O-bis (trimethylsilyl)trifluoroacetamide (BSTFA), squalene and c-tocopherol from Sigma–Aldrich (Seelze, Germany); K2HPO4 and KH2PO4 from Serva (Heidelberg, Germany); amygdalin, benzoic acid, chlorogenic acid, 3,4-dihydroxybenzoic acid, 3,5-dimethoxycinnamaldehyde, gallic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, b-sitosterol, syringaldehyde, syringic acid, vanillin, vanillic acid and rutin from Extrasynthese (Lyon-Genay, France); neochlorogenic acid from TransMIT GmbH (Marburg, Germany) and 2,20 -azobis(2-amidinopropane) dihydrochloride (AAPH) from Wako Chemicals (Neuss, Germany). 2.2. Plant material Three different parts of the Lorraine fruits (5 kg of which were purchased from a local supermarket in the middle of August 2004, ATAC, Metz, France) were studied, namely the peels, flesh and pits after separation and lyophilisation. The lyophilised samples were ground to a fine homogeneous powder prior to extraction. 2.3. Extraction protocol

Table 3c Amounts of phenolic compounds in the pit extract of Prunus domestica L. as determined by reverse-phase analytical HPLC–ESI-MS. Identifier in Fig. 2

Compound

Amount (mg/kg)

I II III IV V VI VII VIII IX X XI

Benzoic acid* p-Hydroxybenzaldehyde* p-Hydroxybenzoic acid* Vanillin 3,4-Dihydroxybenzoic acid Vanillic acid Gallic acid* Syringaldehyde* Syringic acid* Coniferyl aldehyde 3,5Dimethoxycinnamaldehyde Vanillin diglucoside Amygdalin Dehydro-diconiferyl aldehyde Guajacyl-glycerin-coniferyl aldehyde-1 Guajacyl-glycerin-coniferyl aldehyde-2

1.86 0.15 0.26 102 27 29 0.10 0.41 0.63 11 9

XII XVIII XIX XX XXI Total (g) Total (peel + flesh + pits) in Mirabelle fruits (g) *

48 3791 52 33 54 4.159 5.338

Identified and quantitated by GC–MS.

and phenolic antioxidant profiles in the various botanical parts of the Mirabelle fruit. Furthermore, the antioxidant capacity in a range of in vitro assays of the total extracts and major individual compounds was also evaluated. 2. Materials and methods 2.1. Chemicals and reagents Acetic acid, acetonitrile, dichloromethane, ethyl acetate, ethylenediamine tetraacetic acid, hypoxanthine, uric acid, xanthine, xanthine oxidase [EC 1.1.3.22], salicylic acid, sodium hydrogen phosphate and anhydrous sodium sulphate were obtained from E. Merck (Darmstadt, Germany); FeSO47H2O and 2,4,6,-tripyridyl-s-triazine complex (TPTZ) from Riedel–deHaen (Seelze, Germany); coniferyl aldehyde, DPPH (1,1-diphenyl-2-picrylhydrazyl),

The dried peels, flesh and pits (5 g of each) were extracted (3 h) with methanol (150 ml) following delipidation with hexane in a Soxhlet apparatus. These extracts were dissolved in methanol (5.0 ml) prior to analytical reverse-phase HPLC–ESI-MS and GC– MS. 2.4. Analytical HPLC Analytical HPLC was conducted on a Hewlett–Packard (HP) 1090 liquid chromatography (Agilent Technologies, Waldbronn, Germany) fitted with a C18, reverse-phase (5 lm) column (25 cm  4 mm I.D.; Latek, Eppelheim, Germany) as described previously (Khallouki et al., 2007; Owen, Giacosa, et al., 2000). Briefly, samples of Mirabelle plum extracts were dissolved in methanol (5.0 ml) and, when necessary, further diluted prior to injection (20 ll) into the HPLC. The mobile phase consisted of 2% acetic acid in water (solvent A) and methanol (solvent B) with the following gradient: 95% A for 2 min, to 75% A in 8 min, to 60% A in 10 min, to 50% A in 10 min and 0% A until completion of the run. The flow rate was 1 ml/min. Phenolic compounds in the eluate were detected at 250, 278 and 340 nm with a diode array UV detector (HP 1040 M). 2.5. Semi-preparative HPLC Semi-preparative HPLC was conducted on a HP 1100 liquid chromatography (Agilent Technologies, Waldbronn, Germany) fitted with a similar C18 column (10 mm I.D.) as described for analytical HPLC (Owen, Mier, Giacosa, Spiegelhalder, et al., 2000). Briefly, for the separation of individual compounds in the extracts, the mobile phase (3 ml/min) consisted of 0.2% acetic acid in water (solvent A) and acetonitrile (solvent B), utilising the following solvent gradient profile over a total run time of 50 min: initially 95% A for 1 min; reduced to 90% A over 9 min; to 85% A over 10 min; to 80% A over 10 min; to 0% A over 5 min and continuing at 0% A until completion of the run. Peaks eluting from the column were collected on a HP 220 Microplate Sampler and subsequently lyophilised. 2.6. Electrospray ionisation mass spectrometry (ESI-MS) HPLC–ESI-MS was conducted on an Agilent 1100 HPLC, coupled to an Agilent single-quadrupole, mass-selective detector (HP 1101;

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CHO -H

H

HOH2C

O HO

+

CHO

C H

C

H

H 2C

O

OCH3 OCH3

m/z = 355.1

CHO

H

HOH2C

C

-H+

-

C H

OCH3

HO

OCH3

C

-H+

m/z = 337.1

-

C H

-H+ CHO

H

H 2C

O

C

-

-H+ CHO

C H

H

H 2C

C

-

C H

+

HO OCH3

O m/z = 325.2 CHO

H

HOH2C

C

-H+

-

C H

O

HO

O HO

OCH3

OCH3

m/z = 322.1

m/z = 307.1

O HO

m/z = 295.1 Fig. 6. Nano-ESI-MS-MS fragmentation scheme of dehydro-diconiferyl aldehyde (19) in the negative ion mode.

Agilent Technologies, Waldbronn, Germany), whereas nano-ESIMS-MS was performed with purified samples, following semi-preparative HPLC, dissolved in methanol on a Finnigan MAT TSQ-7000 triple-quadrupole mass spectrometer (Finnigan, San Jose, California, USA) equipped with a nanoelectrospray source (EMBL, Heidelberg, Germany) using both the positive- and negative-ion modes (Pfundstein et al., 2010).

with the appropriate molecular weight correction. Guajacyl-glycerin-coniferyl aldehyde isomer-1, guajacyl-glycerin-coniferyl aldehyde isomer-2 and dehydro-diconiferyl aldehyde were quantified from standard curves of the respective compounds following purification by semi-preparative HPLC. Benzoic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, gallic acid, syringaldehyde and syringic acid were calculated from standard curves of authentic standards (0–50 lg/ml) as analysed by GC–MS.

2.7. Gas-chromatography–mass spectrometry (GC–MS) 2.9. Antioxidant assays Analyses were performed using a HP 5973 mass spectrometer coupled to a HP 6890 gas chromatography. Separation of the analytes was achieved using a HP 5MS capillary column, 30 m  0.25 mm I.D., 0.25 lm film thickness. Helium was used as carrier gas with a linear velocity of 0.9 ml/s. Sample aliquots of 1 ll were injected. The oven temperature programme was as follows: initial temperature 100 °C, 100–270 °C at 4 °C/min, 20 min at 270 °C. The GC injector temperature was 250 °C; the transfer line temperature was held at 280 °C. The conditions used in the EI mode were as follows: ion source temperature, 230 °C: electron energy, 70 eV: filament current, 34.6 lA and electron multiplier voltage, 1200 V. Prior to GC–MS, dried methanolic extracts were derivatised by addition of 100 ll of BSTFA at 30 °C for 30 min.

The HPLC-based hypoxanthine/xanthine oxidase antioxidant assay was conducted exactly as described previously (Owen, Mier, Giacosa, Hull, et al., 2000), whereas the DPPH, FRAP and ORAC assays were conducted as described by Pfundstein et al. (2010). 2.10. Statistics IC50 values were determined using the Table Curve programme (Jandel Scientific, Chicago, IL, USA). 3. Results

2.8. Quantitation of phenolic compounds

3.1. General characteristics of Prunus  domestica L. (Mirabelle)

Amygdalin, chlorogenic acid, coniferyl aldehyde, 3,4-dihydroxybenzoic acid, 3,5-dimethoxycinnamaldehyde, neochlorogenic acid, vanillin, vanillic acid and rutin were quantitated against standard curves of authentic standards (0–4 mM) as analysed by analytical HPLC. Cryptochlorogenic acid and cis-chlorogenic acid were calculated against the standard curve of chlorogenic acid, while vanillin diglucoside was calculated versus the standard curve of vanillin

Twenty Mirabelle plums were separated into peel, flesh and pits. The wet and dry weights were, respectively, peel (35.48 v. 9.76 g); flesh (142.37 v. 30.90 g) and pits (11.20 v. 8.67 g). The yield of oil in the hexane extracts from the flesh was (3.82%) and pits (4.06%), while the peel yielded a waxy material (16.26%). The yield of the methanol extracts (from 5 g dry material) was peel (4.58 g; 92%), flesh (4.51 g; 90%) and pits (224 mg; 4.5%).

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H HOH2C

C

C

-H+ CHO

H

H H 2C

O OH

OCH3

OCH3

OCH3

H OH

C

C

HO

m/z = 354.9

-H CHO

+

-

H 2C

H

C

C

-H+ CHO

-

H

O OH

HO

OCH3

OCH3

m/z = 195.0

H

OCH3

H

-

+

C

-

HO

m/z = 373.1

+ HOH2C -H

C

-H+ CHO

O OH

HO

HO

-

OCH3

m/z = 325.0

m/z = 176.8

+ HOH2C -H

-

OH HO m/z = 164.9 Fig. 7. Nano-ESI-MS-MS fragmentation scheme of guajacyl-glycerin-coniferyl aldehyde-1 (20) in the negative ion mode.

3.2. Oil As an example, a GC–MS chromatogram of the oil obtained from the flesh by hexane extraction of P. domestica L. (Mirabelle) is depicted in Fig. 1a. The free fatty acids detected were palmitoleic acid (0.58%), palmitic acid (9.26%), linoleic acid (28.70%), oleic acid (59.26%) and stearic acid (2.20%). Low amounts (identified only) of diacylglycerols, c-tocopherol and b-sitosterol were also detectable (Fig. 1b).

3.3. Methanol extracts The structures of the phenolic compounds detected and identified in Mirabelle fruits are depicted in Fig. 2, whereas the nano-ESIMS-MS and GC–MS data are listed in Tables 1 and 2, respectively. Reverse-phase analytical HPLC chromatograms of the methanolic extracts of the peels, flesh and pits of P. domestica L. (Mirabelle) are presented in Fig. 3–5. The total content of phenolic antioxidants in the whole fruits was 5.338 g/kg with 456 mg/kg (9%; Table 3a), 701 mg/kg (13%; Table 3b) and 4159 mg/kg (78%; Table 3c) detected in the peels, flesh and pits respectively (see Figs. 6 and 7).

3.3.1. Peels The major components detected in the peels (Fig. 3a) were 3,4dihydroxybenzoic acid (270 mg/kg) and rutin (186 mg/kg) representing 90% of a low level of phenolic compounds in this botanical part of Mirabelle.

Table 4 Antioxidation assays: comparison of methanol extracts from the various botanical parts of Prunus domestica L. Extract

HX/XO IC50 (mg/mL)a

DPPH IC50 (mg/mL)b

FRAP EC1 (mg/mL)c

ORAC (units)d

Peels Flesh Pits

4.36 2.88 0.79

2.81 0.93 0.33

62.5 38.5 3.9

0.31 0.86 21.03

a IC50: concentration of extract (mg/mL) where DHBA production is reduced by 50%. b IC50: concentration of extract (mg/mL) where 50% of the DPPH radical is scavenged. c EC1: concentration of extract (mg/mL) giving an absorbance increase equivalent to 1 mM Fe(II) solution. d 1 ORAC unit equals the inhibition of the declining fluorescence produced by 1 lM Trolox.

3.3.2. Flesh The major components in the flesh (Fig. 3b) were the echinoids, of which the proportion of neochlorogenic acid (575 mg/kg including cis-neochlorogenic acid, 9 mg/kg), cryptochlorogenic acid (22 mg/kg) and chlorogenic acid (94 mg/kg) was 83:3:14 respectively along with 5-O-p-coumaroyl quinate (32 mg/kg). 3.3.3. Pits The major components of the pith extracts (Fig. 3c) quantitated by HPLC–ESI-MS were amygdalin (3.791 g/kg) along with vanillin (102 mg/g), a neolignan dehydro-diconiferyl aldehyde (52 mg/kg) plus two guajacyl-glycerin-coniferyl aldehyde isomers (33 and

F. Khallouki et al. / Food Chemistry 133 (2012) 697–706 Table 5 Antioxidation assays: comparison of pure polyphenolic compounds isolated from the various botanical parts of Prunus domestica L. Phenolic compound

HX/XO IC50 (mM)a

DPPH IC50 (lM)b

FRAP EC1 (lM)c

ORAC (units)d

Gallic acid⁄

e Prooxidant 0.94 3.62 >4.00 3.52 4.19

3.3

232

1.18

6.7 10.1 10.5 11.0 18.8

484 382 478 527 448

3.75 1.34 3.07 2.42 2.22

3.34 2.03 2.25

22.4 67.7 1015

4.34 1.91 1.62

Vanillic acid

2.67

1838

Vanillin

2.54

5682

>4.00 1.32 2.75 2.95

Inactive Inactive Inactive Inactive

532 470 Mild activity Mild activity Mild activity Inactive Inactive Inactive Inactive

Rutin Syringic acid⁄ Chlorogenic acid Neo-chlorogenic acid 3,5-Dimethoxy-4cinnamaldehyde 3,4-DHBA Coniferyl aldehyde Syringaldehyde⁄



p-HBAL p-HBA⁄ Benzoic acid⁄ Amygdalin

2.60 2.94 1.17 4.15 0.01 0.02

3,4-DHBA = 3,4-dihydroxybenzoic acid. p-HBAL = p-hydroxybenzaldehyde. pHBA = p-hydroxybenzoic acid. Due to their low concentration, as identified by GC– MS antioxidant assays were conducted using authentic reference standards. a IC50: concentration of substance (mM) where DHBA production is reduced by 50%. b IC50: concentration of substance (lM) where 50% of the DPPH radical is scavenged. c EC1: concentration of substance (lM) giving an absorbance increase equivalent to 1 mM Fe(II) solution. d 1 ORAC unit equals the inhibition of the declining fluorescence produced by 1 lM Trolox. e At 4 mM, increase in DHBA = 56%. * Due to their low concentration, as identified by GC-MS antioxidant assays were conducted using authentic reference standards.

54 mg/kg), vanillyl diglucoside (48 mg/kg), vanillic acid (29 mg/ kg), 3,4-dihydroxybenzoic acid (27 mg/kg), coniferyl aldehyde (11 mg/kg), and 3,5-dimethoxycinnamaldehyde (9 mg/kg). The following minor phenolic components were also identified and quantitated by GC–MS namely, benzoic acid (1.86 mg/kg), syringic acid (0.63 mg/kg), syringaldehyde (0.41 mg/kg), 4-hydroxybenzoic acid (0.26 mg/kg), 4-hydroxybenzaldehyde (0.15 mg/kg) and gallic acid (0.10 mg/kg). Amygdalin represented over 90% of the phenolic compounds present in the pits. 3.4. Antioxidant assays The antioxidant capacities (Table 4) measured in the methanol extracts of the peels, flesh and pits reflected their content of total phenolic compounds with efficacy increasing of the order peels < flesh < pits across the assays. Of the purified phenolic compounds tested (Table 5) across the range of in vitro antioxidant assays, rutin was superior (rank-1) followed by 3,4-dihydroxybenzoic acid and chlorogenic acid (rank-2), syringic acid (rank-3) and neochlorogenic acid (rank-4). This was obtained by ranking their activity in each assay from highest activity to lowest activity. The mean for the 4 assays was then taken. Amygdalin, the major phenolic component of Mirabelle fruits, which has a chartered history in terms of cancer chemoprevention was totally inactive in the DPPH, FRAP and ORAC assays and ranked only 8th of the fifteen compounds tested in the HPLC-based HX/XO assay. 4. Discussion The only phenolic compounds detected in the peels (Fig. 3a) of P. domestica L. were 3,4-dihydroxybenzoic acid and rutin (quercetin-

705

3-rutinoside), whereas the major components detected in the flesh (Fig. 3b) were the echinoids dominated by neochlorogenic acid giving a total of 723 mg/kg (on a dry weight basis), which is similar to that reported (629 mg/kg) by Herrmann (1989). The ratio of echinoids detected was also very similar to that reported by Herrmann (1989) in plum fruits at 83:3:14 versus 86:1:12, but slightly discordant with that reported by Nakatani et al. (2000) in prunes at 78:18:4, for 3-CQA, 4-CQA and 5-CQA, respectively. The major reason for these differences is probably that Herrmann (1989), Nakatani et al. (2000), Kikuzaki et al. (2004), Nunes et al. (2008), Slimestad et al. (2009) and Kosar et al. (2010) studied P. domestica L., which is known as the common plum. In this work we describe a subspecies cross and hybrid variety (there are several other forms), known as Mirabelle plums from the Lorraine region of France, Saarbrüken (Germany) and Luxembourg and are differentiated from P. domestica L. by their colour (yellow) and smaller size. Our data are, however, in contrast to that of Bouayed, Rammal, Dicko, Younos, and Soulimani (2007), who reported chlorogenic acid and not neochlorogenic acid as the main echinoid in Mirabelle. The pits (Fig. 3c) however displayed by far the more complex profile of phenolic antioxidants. In total, 16 individual phenolic compounds have been identified thus far in methanol extracts of Prunus pits. The major components identified and quantitated by HPLC–ESI-MS in pits comprise amygdalin, vanillin, coniferyl aldehyde, 3,5-dimethoxycinnamaldehyde along with a neolignan, dehydro-diconiferyl aldehyde, plus two guajacyl-glycerin-coniferyl aldehyde isomers. Lesser amounts of 3,4-dihydroxybenzoic acid, vanillin diglucoside and vanillic acid were also identified by HPLC–ESI-MS, whereas the following were detected by GC–MS in very minor amounts namely benzoic acid, syringic acid, syringaldehyde, 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde and gallic acid. This represents the first report of phenolic compounds in the pits of Mirabelle fruits and indeed of those identified, only 3,4dihydroxybenzoic acid has previously been described in this fruit. The antioxidant capacities of the various raw extracts corresponded well with the overall content of phenolic contents therein, whereby the extracts of the pits were far more effective across the antioxidant assays than those of the flesh and peels, respectively. As previously shown the major components identified in fruit (peel + flesh) comprise 3,4-dihydroxybenzoic acid, rutin and the echinoids; all of which show relatively high antioxidant capacities in vitro (Table 5). Therefore consumption of Mirabelle represents a rich source of antioxidants in the western diet, either as a seasonally fresh fruit, or else as prunes during the remainder of the year. Obviously the pits are not generally consumed, but with their high content and variability of phenolic compounds they represent a waste product which could be utilised in the future for the extraction of natural food preserving antioxidants. However, it should be noted that a considerable amount of amygdalin was detected in the pith extracts. This compound has a chartered history in terms of cancer therapeutics. It was first detected in the pits of apricots in 1830 and was subsequently used in the clinical domain (Russia, ca. 1845; USA, 1920s) as an anti-cancer drug with equivocal success. A major problem is that administration of amygdalin as a chemotherapeutic agent results, after hydrolysis by body enzymes, in the production of hydrogen cyanide and this can reach lethal levels in the blood after continuous administration. The data presented in this paper is the first to report on the content of phenolic antioxidants in the various botanical parts of Mirabelle fruits and shows that both the peel and flesh of Mirabelle fruits contain significant amounts of potentially cancer chemopreventive agents, based on a range of in vitro assays (Tables 4 and 5). Of the whole Mirabelle fruits however, the pits as waste products contain the highest amount of phenolic antioxidants (82%), but these are dominated by amygdalin (Table 3c), a nitrogenous

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compound which has been banned by the FDA as a cancer chemotherapeutic agent. If the pits of Prunus are to be used as a potential source of cheap antioxidants, it is therefore recommended that amygdalin is removed. References Arjmandi, B. H., Khalil, D. A., Lucas, E. A., Georgis, A., Stoecher, B. J., Hardin, C., et al. (2002). Dried plums improve indices of bone formation in postmenopausal women. Journal of Women’s Health and Gender-Based Medicine, 11, 61–68. Bouayed, J., Rammal, H., Dicko, A., Younos, C., & Soulimani, R. (2007). Chlorogenic acid, a polyphenol from Prunus domestica (Mirabelle), with coupled anxiolytic and antioxidant effects. Journal of Neurological Science, 262, 77–84. Cao, G., Booth, L. H., Sadowski, J. A., & Prior, R. L. (1998). Increases in human plasma antioxidant capacity after consumption of controlled diets high in fruit and vegetables. American Journal of Clinical Nutrition, 68, 1081–1087. Chopra, R. N., Nayar, S. C., & Chopra, I. C. (1956). Glossary of Indian medical plants. New Delhi, India: C.S.I.R., p. 205. Edenharder, R., Kurz, P., John, K., Burgard, S., & Seeger, K. (1994). In vitro effect of vegetable and fruits juices on the mutagenicity of 2-amino-3-methylimidazo [4,5-f] quinolone. In vitro effect of vegetable and fruit juices on the mutagenecity of 2-amino-3-methylimidazo [4,5,-f] quinoline and 2-amino3,4-dimethylimidazo[4,5,-f] quinoxaline. Food and Chemical Toxicology, 32, 443–459. Fujii, T., Ikami, T., Xu, J. W., & Ikeda, K. (2006). Prune extract (Prunus domestica L.) Suppresses the proliferation and induces the apoptosis of human colon carcinoma Caco-2. Journal of Nutrition Science and Vitaminology, 52, 389–391. Henning, W., & Herrmann, K. (1980). Flavonol glycosides of plums of the species Prunus domestica L. and Prunus salicina Lindley. Zeitschrift für LebensmittelUntersuchung and Forschung, A, 171, 111–118. Herrmann, K. (1989). Occurrence and content of hydoxycinnamic and hydroxybenzoic acid compounds in foods. Critical Review of Food Science and Nutrition, 28, 315–347. Kahlon, T. S., & Smith, G. E. (2007). In vitro binding of bile acids by blueberries, plum, strawberries cherries, cranberries and apples. Food Chemistry, 100, 1182–1187. Kayano, S. I., Yamada, N. F., Suzuki, T., Ikami, T., Shioaki, K., Kikuzaki, H., et al. (2003). Quantitative evaluation of antioxidant components in Prunes (Prunus domestica L.). Journal of Agricultural and Food Chemistry, 51, 1480–1485. Khallouki, F., Younos, C., Soulimani, R., Oster, T., Charrouf, Z., Spiegelhalder, B., et al. (2003). Consumption of argan oil (Morocco) with its unique profile of fatty acids, tocopherols, squalene, sterols and phenolic compounds should confer valuable cancer chemopreventive effects. European Journal of Cancer Prevention, 12, 67–75. Khallouki, F., Haubner, R., Hull, W. E., Erben, G., Spiegelhalder, B., Bartsch, H., et al. (2007). Isolation, purification and identification of ellagic acid, catechins and procyanidin derivatives from the root bark of Anisophyllea dichostyla R. Br. Food and Chemical Toxicology, 45, 472–485. Kikuzaki, H., Kayano, S., Fukutsuka, N., Aoki, A., Kasamatsu, K., Yamasaki, Y., et al. (2004). Abscisic acid related compounds and lignans in prunes (Prunus domestica L.) and their oxygen radical absorbance capacity (ORAC). Journal of Agricultural and Food Chemistry, 52, 344–349.

Kono, Y., Kobayashi, K., Tagawa, S., Adachi, K., Ueda, A., Sawa, Y., et al. (1997). Antioxidant activity of polyphenolics in diets. Rate constants of reactions of chlorogenic acid and caffeic acid with reactive species of oxygen and nitrogen. Biochimica, Biophysica Acta, 1335, 335–342. Kosar, S., Fatima, I., Mahmood, A., Ahmed, R., Malik, A., Talib, S., et al. (2010). Purunusides A–C, alpha-glucosidase inhibitory homoisoflavone glucosides from Prunus domestica. Archives of Pharmaceutical Research, 32, 1705–1710. Nakatani, N., Kayano, S., Kikuzaki, H., Sumino, K., Katagiri, K., & Mitani, T. (2000). Identification, quantitative determination, and antioxidative activities of chlorogenic acid isomers in prune (Prunus domestica L.). Journal of Agricultural and Food Chemistry, 48, 5512–5516. Nunes, C., Guyot, S., Marnet, N., Barros, A. S., Saraiva, J. A., Renard, C. M., et al. (2008). Characterization of plum procyanidins by thiolytic depolymerisation. Journal of Agricultural and Food Chemistry, 56, 5188–5196. Owen, R. W., Giacosa, A., Hull, W. E., Haubner, R., Würtele, G., Spiegelhalder, B., et al. (2000). Olive-oil consumption and health: The possible role of antioxidants. Lancet Oncology, 1, 107–112. Owen, R. W., Mier, W., Giacosa, A., Hull, W. E., Spiegelhalder, B., & Bartsch, H. (2000). Phenolic compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols, simple phenols, secoiridoids, lignans and squalene. Food and Chemical Toxicology, 38, 647–659. Owen, R. W., Mier, W., Giacosa, A., Spiegelhalder, B., Hull, W. E., & Bartsch, H. (2000). Phenolic and lipid components of olive oils: identification of lignans as major components of the phenolic fraction of olive oil. Clinical Chemistry, 46, 976– 988. Pfundstein, B., El Desouky, S. K., Hull, W. E., Haubner, R., Erben, G., & Owen, R. W. (2010). Polyphenolic compounds in Egyptian medicinal plants (Terminalia bellerica, Terminalia chebula and Terminalia horrida): Characterization, quantitation and determination of antioxidant capacities. Phytochemistry, 71, 1132–1148. Raynal, J., Moutounet, M., & Souquet, J. M. (1989). Intervention of phenolic compounds in plum technology. 1. Changes during drying. Journal of Agricultural and Food Chemistry, 37, 1046–1050. Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20, 933–956. Slimestad, R., Vangdal, E., & Brede, C. (2009). Analysis of phenolic compounds in six Norwegian plum cultivars (Prunus domestica L.). Journal of Agricultural and Food Chemistry, 57, 11370–11375. Stacewicz-Sapuntzakis, M., Bowen, P. E., Hussain, E. A., Damayanti-Wood, B. I., & Farnsworth, N. R. (2001). Chemical composition and potential health effects of prunes: A functional food? Critical Review of Food Science and Nutrition, 41, 251–286. Tinker, L. F., Davis, P. A., & Schneeman, B. O. (1994). Prune fiber or pectin compared with cellulose lowers plasma and liver lipids in rats with diet-induced hyperlipidemia. Journal of Nutrition, 124, 31–40. Usenika, V., Štampar, F., & Vebericˇ, R. (2009). Anthocyanins and fruit colour in plums (Prunus domestica L.) during ripening. Food Chemistry, 114, 529–534. Yu, M. H., HyoGwon, I., Syng-ook, L., Chang, S., Park, D. C., & Inseon, L. (2007). Induction of apoptosis by immature fruits of Prunus salicina Lindl. cv. Soldam in MDA-MB-231 human breast cancer cells. International Journal of Food Science and Nutrition, 58, 42–53.