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Identification and Characterisation of Anthocyanins from Wild Mulberry (Morus Nigra L.) Growing in Brazil N. M.A. Hassimotto, M. I. Genovese and F. M. Lajolo Food Science and Technology International 2007; 13; 17 DOI: 10.1177/1082013207075602 The online version of this article can be found at: http://fst.sagepub.com/cgi/content/abstract/13/1/17

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Identification and Characterisation of Anthocyanins from Wild Mulberry (Morus Nigra L.) Growing in Brazil N.M.A. Hassimotto, M.I. Genovese and F.M. Lajolo* Laboratório de Química, Bioquímica e Biologia Molecular de Alimentos, Departamento de Alimentos e Nutrição Experimental, FCF, Universidade de São Paulo, Av. Prof. Lineu Prestes 580, Bloco 14, 05508-900 São Paulo, SP, Brazil Anthocyanin pigments from the wild mulberry (Morus nigra L.) growing in Brazil, were extracted with methanol/water/acetic acid (70 : 30 : 5 v/v), and the crude methanolic extract (CME) was cleaned up by polyamid solid-phase extraction (SPE). Two major anthocyanins were isolated by paper chromatography, and their chemical structures were identified by spectral analyses, HPLC/DAD and mass spectroscopy. Information from HPLC profiles, saponification and acid hydrolysis of the mulberry anthocyanins showed that the two major pigments were cyanidin-3-glucoside (ca. 71% of the total) and cyanidin-3-glucosylrhamnoside (ca. 19% of the total), with no cinnamoyl groups. The CME (at 10 M gallic acid equivalent), neutral flavonoids (NF) and acidic flavonoids (AF) eluate from SPE (at 1 M GAE), showed a higher antioxidant activity than quercetin and synthetic antioxidant (butyrate hydroxytoluene (BHT)) at 10 M, by the -carotene bleaching method. Key Words: mulberry, anthocyanins, ESI-MS/MS, spectral analysis

INTRODUCTION Anthocyanins are a group of naturally occurring phenolic compounds responsible for the colour of many plants, flowers and fruits. They are the glycosylated form of anthocyanidins, polyhydroxy and polymethoxy derivates of flavylium salts. Among them, the most common are cyanidin (cy), pelargonidin, delphinidin, peonidin, petunidin and malvidin (Clifford, 2000). Recently, a great deal of renewed interest in anthocyanins has emerged because of their potential health benefits as antioxidant and anti-inflammatory agents (Kähkönen and Heinonen, 2003). The antiinflammatory activity is related to their ability to inhibit the two isoforms of the cyclooxygenase enzyme, COX-1 and COX-2, as demonstrated for the anthocyanins isolated from raspberries and sweet cherries

*To whom correspondence should be sent (e-mail: [email protected]). Received 21 December 2005; revised 26 June 2006. Food Sci Tech Int 2007; 13(1):17–25 © 2007 SAGE Publications ISSN: 1082-0132 DOI: 10.1177/1082013207075602

(cyanidin-3-glucosylrutinoside and cyanidin-3-rutinoside) at 250 M (Seeram et al., 2001) and also for the anthocyanidins, cy and malvidin at 80 M. The inhibitory activities were comparable to the commercial anti-inflammatory drugs ibuprofen and naproxen, at 10 M concentration (Seeram et al., 2003). In addition, the anthocyanin extracted from tart was able to reduce paw edema induced by intraplantar injection of 1% carragenan in rats (Tall et al., 2004). Anthocyanins, as other flavonoid classes, present high antioxidant activity in several oxidation systems, related to their ability to inhibit lipid peroxidation, radical scavenging activity and metal chelating properties (Harborne and Williams, 2000). Studies on the relationship between anthocyanin structure and antioxidant activity have shown that different patterns of hydroxylation and glycosylation in anthocyanidins appear to modulate their antioxidant properties. Among common anthocyanidins, cy and delphinidin, which present 3and 4-hydroxyls in the backbone, showed the highest value of trolox equivalent antioxidant activity (TEAC) in the ABTS system (Rice-Evans et al., 1996), while malvidin was the best oxidation inhibitor in liposome and LDL oxidation systems catalysed by cupper (Satué-Gracia et al., 1997). The pattern of glycosylation also affects the antioxidant activity. While Rice-Evans et


N.M.A. HASSIMOTTO ET AL.

18

al. (1996) have shown a general trend of decreasing TEAC by glycosylation, Wang et al. (1996) showed that cyanidin-3-glucoside had the highest ORAC activity among the anthocyanins tested, higher than those of the aglycon forms. Further, fruit extracts containing high levels of anthocyanins, such as those from strawberry and plum, demonstrated high radical scavenging activity by the DPPH method (Espin et al., 2000), and Rubus species present high TEAC and ferric reducing antioxidant power (FRAP) (Deighton et al., 2000). In addition, direct correlation between antioxidant activity and anthocyanin content was demonstrated for Vaccinun species (Prior et al., 1998) and blueberry, strawberry and raspberry (Kalt et al., 1999). Among dietetic sources, anthocyanins are especially abundant in berries, such as bilberry (Vaccinium myrtillus), blackberry (Vaccinium vitis-idaea) and cowberry (Ribes nigrum) with 6,000, 2,360 and 680 mg/kg, respectively (Kähkönen et al., 2003). In Rubus species the anthocyanin varies from 0.1 to 1.186 g/kg f.w. (Deighton et al., 2000) and from 150 to 300 mg/kg (f.w.) for three cultivars of strawberry grown in Brazil (Cordenunsi et al., 2003). Morus nigra L. is a rustic plant growing wildly, cultivated in gardens or commonly used in sericulture. The fresh fruit is hardly commercialised because it has a fragile structure and low stability to storage, being generally processed as jelly or juice. The most known species are white mulberry (Morus alba), black mulberry (Morus nigra), and red mulberry (Morus rubra), with a large range of colour varying from white to black and red. The first two species are from western Asia and the red mulberry is a native of North America (Holmes, 1996). A previous screening in our laboratory showed that wild mulberry had the highest antioxidant activities among Brazilian fruits, by both the liposome oxidation and the -carotene bleaching methods (Hassimotto et al., 2005). These results justified a more detailed study of the compounds involved in this activity. Accordingly, the objectives of this work were to determine the content and profile of flavonoids of wild mulberry grown in Brazil and to characterise their anthocyanins, in order to identify possible components with a high potential to promote human health.

MATERIALS AND METHODS Material Fruit Three batches per harvest, each consisting of 2 kg of ripe wild mulberry (Morus nigra L.), were harvested from a house garden situated in São Paulo city (Brazil), during the fruit-bearing season (from September to October in 2003 and 2004). Undamaged and

completely ripe (uniform purple colour) berries were washed with tap water and immediately frozen in liquid nitrogen and stored at 70 °C until the time of the analysis. Prior to the analysis, the frozen samples were ground and homogenised under liquid nitrogen. Chemicals Bis(trimethylsylil)tri-fluoroacetamide, methoxyamine, -carotene, linoleic acid, Tween 40, BHT, quercetin, kaempherol and clorogenic acid were purchased from Sigma Chemical Co. (St Louis, USA). The anthocyanidins cy and pelargonidin were obtained from Extrasynthèse (Genay, France). Thiobarbituric acid was obtained from Merck Chemical Co. (Darmstadt, Germany). All others reagents used were analytical or HPLC grade. Methods Flavonoid Extraction It was performed in duplicate according to the method of Price et al. (1999) with slight modifications. The sample (5 g) was extracted three times in 100 mL of methanol/water/acetic acid (70 : 30 : 5 v/v) for 2 min (Brinkmann homogeniser, Polytron-Kinematica GmbH, Kriens-Luzern, Sweden) in an ice bath. The homogenate was filtered under reduced pressure through filter paper (Whatman No. 06). The extract was concentrated until methanol elimination under vacuum at 40 °C on a rotary evaporator (Rotavapor RE 120, Büchi, Flawil, Sweden) and made up to 50 mL with distilled water for application to SPE columns. SPE An aliquot (3.5mL) of the extract was passed through polyamide (CC-6, Macherey-Nagel, Germany) columns (1g/6mL) previously conditioned with 20mL of methanol and 60mL of distilled water. Impurities were washed out with 20mL of distilled water and retained flavonoids were eluted with methanol (50mL), to elute NF, followed by methanol: ammonia (99.5:0.5v/v), to elute AF (Price et al, 1999). The flow rate through the columns was controlled by means of a vacuum manifold Visiprep 24DL (Supelco, Bellefonte, PA). Each elute was evaporated to dryness under reduced pressure at 40°C, dissolved in methanol: acetic acid (99:5v/v), and filtered through a 0.22m tetrafluoroethylene (PTFE) filter (Milipore Ltd, Bedford, MA) prior to HPLC analysis. Flavonoid Determination by HPLC Flavonoids were determined by reversed-phase HPLC in a Hewlett-Packard 1100 system with auto sampler and quaternary pump coupled to a diode array


Anthocyanins from Wild Mulberry (Morus Nigra L.) detector (DAD). The column used was a Prodigy 5 m ODS (3) reversed-phase silica (250 mm 4.6 mm i.d., Phenomenex Ltd). The solvents were: (A) water/ tetrahydrofuran/trifluoroacetic acid (98 : 2 : 0.1 v/v) and (B) acetonitrile. For NF, the solvent gradient consisted of 8% B at the beginning, 10% at 5 min, 17% at 10 min, 25% at 15 min, 50% at 25 min, 90% at 30 min, 50% at 32 min, 8% at 35 min (run time, 35 min). For the AF, the solvent gradient was the same (run time, 35 min) as that used by Price et al. (1999). Eluates were monitored at 270, 370 and 525 nm. Flow rate was 1 mL/min, column temperature was 30 °C and injection volume was 5–20 L. Samples were injected in triplicate. Flavonoids were quantified using external standards. Peak identification was performed by comparison of retention time and spectra with the standards and the library spectra. Results were expressed as milligrams of aglycon per 100 g of fresh weight (f.w.). Anthocyanin Purification Purification was performed by descending paper chromatography according to the methods described by Harborne (1967). NF from SPE were streaked on Whatman 3MM and chromatographed in 1% HCl in water. Bands were cut off, eluted with methanol: acetic acid (95 : 5 v/v) and evaporated to dryness under reduced pressure at 40 °C in a rotary evaporator. The pigments were dissolved in methanol : acetic acid (95 : 5 v/v), re-chromatographed in 15% acetic acid in water, and eluted in the same way. The purified pigments were dissolved in 0.01% HCl in methanol to UV-V spectrophotometric characterisation and mass–mass spectroscopy. Alkaline Hydrolysis of Anthocyanins Alkaline hydrolysis was done according to the method of Ordaz-Galindo et al. (1999). An aliquot of the purified pigments of mulberry was evaporated to dryness under nitrogen and dissolved in 10 mL of 10% aqueous KOH in a screw-cap test tube. The tube was flushed with nitrogen and capped. The pigments were hydrolysed for 8 min at room temperature in the dark. The pigments were reconverted to their red flavilium salt form by the addition of 2 N HCl, then adsorbed onto CC-6 polyamide Sep-Pak cartridge, washed with water, eluted with 0.01% HCl in methanol, and evaporated to dryness under reduced pressure at 40 °C. The pigments were dissolved in methanol: acetic acid (95 : 5 v/v) for HPLC analyses and 0.01% HCl in methanol for UV-V spectrophotometric characterisation. Acid Hydrolysis of Anthocyanins

19

ments of mulberry was evaporated to dryness under reduced pressure at 40 °C and dissolved with 10 mL of 2 N HCl in a screw-cap test tube. The pigment was hydrolysed for 45 min at 100 °C in a water bath, under nitrogen flux. The hydrolysate was purified using CC-6 polyamide Sep-Pak cartridge, washed with water, eluted with 0.01% HCl in methanol, and evaporated to dryness under reduced pressure at 40 °C. The pigments were dissolved in methanol: acetic acid (95 : 5 v/v) for HPLC analyses and 0.01% HCl in methanol for UV-V spectrophotometric characterisation. The washing water was recovered for carbohydrate analyses by gas chromatography coupled to a mass selective detector (GC-MS). UV-V Spectrophotometric Characterisation of Anthocyanins The spectra of the purified pigments were obtained using a Hewlett Packard 8453 spectrophotometer. The pure dry pigments were dissolved in 0.01% HCl in methanol and the solutions were diluted to give an optical density reading in the range from 0.800 to 1.300 at the visible maxima wavelength. The scan wave acquisition was from 200 to 800 nm. Spectral shifts in the presence of aluminium chloride were obtained by adding three drops of a solution of the anhydrous salt in methanol (5%, w/v) onto the cell solution. Gas Chromatography–EI-MS of Carbohydrates The washing water from acidic hydrolysis was neutralised with NaOH and lyophilised. The powder (0.4 g) was mixed with 300 L of methoxamine (20 mg/mL in anhydrous pyridine) and kept at room temperature for 2 h. An aliquot was mixed with methylN-Bis(trimethylsylil)tri-fluoroacetamide (1:1v/v), and heated at 37 °C for 30 min. EI-MS spectra were recorded by coupling a Hewlett Packard 6890 gas chromatograph equipped with a SUPELCO 24181 SPB column (30.0 m 250 m 0.25 m) to a Hewlett Packard 5973 mass selective detector. The operating conditions were as follows: splitless injection (1 L), carrier gas was He at 1.0 mL/min, injector and detector temperature of 300 °C. The column temperature kept at 70 °C for 5 min, and then increased to 300 °C during 10 min, and maintained at this temperature for 50 min. Identifications were based on retention time using standard carbohydrates and comparing the mass spectrum from the NIST library. The mass selective detector conditions were: interface temperature of 250 °C, ion source energy of 70 eV and acquisition full mode scan (from 50 to 500 amV).

Acid hydrolysis was done according to OrdazGalindo et al. (1999). An aliquot of the purified pigDownloaded from http://fst.sagepub.com at UNIV DE SAO PAULO BIBLIOTECA on March 13, 2007 © 2007 SAGE Publications. All rights reserved. Not for commercial use or unauthorized distribution.

20


Electrospray Ionisation (ESI)–Mass Spectrometry (MS/MS) of Anthocyanins Low-resolution MS was done using ESI–MS. The equipment was a Quatro LC, Micromass (Altringham, Cheshire, UK). The MS was performed in positive ion mode. Collision induced dissociation experiments were carried out using argon as the target gas. Pigments 1 and 2 were injected directly into the mass spectrometer. The capillary voltage and temperature were 3.0 KV and 350 °C, respectively. When the molecular ion of the pigment was detected, its MS2 spectrum was obtained using a relative energy of collision of 30 eV.

tion (2 mg/mL in chloroform) was added to a flask containing 40 L of linoleic acid, 1.0 mL chloroform and 0.4 mL of Tween 40 and mixed. The chloroform was evaporated to dryness under nitrogen. After this, oxygenated distilled water (100 mL) was added and the mixture shaken. Aliquots (100 L) of the sample extract were added to 2.9 mL of -carotene solution and mixed. The absorbance of the solution at 470 nm was immediately measured using the spectrometer Hewlett Packard 8453 and after 2 h of incubation in a water bath at 50 °C. The control consisted of 100 L of methanol instead of the sample extract. Antioxidant activity was calculated as percent inhibition relative to the control. The analysis were done in triplicate.

Total Phenolics Were measured in duplicate samples of each extract according to the method of Swain and Hillis (1959), using the Folin-Ciocalteu reagent and gallic acid as standard. The results were expressed as gallic acid equivalents (GAE). In Vitro Antioxidant Assay The antioxidant activity of the extracts obtained from total phenolic analysis was determined according to the -carotene bleaching method described by Miller (1971). An aliquot (20 L) of -carotene solu-

1500

(a)

2

1

3

4

0 Abs (mAU)

(b) 100

6 5

RESULTS AND DISCUSSION HPLC Flavonoids Profile Flavonoids from wild mulberry were separated by HPLC (Figures 1(A) and (B) and identified as two peaks of cy derivates (peaks 1 and 2), one peak of a pelargonidin derivate (peak 3) and one of a quercetin derivative (peak 4), in the methanol elute. On the other side, in the methanol : ammonia elute, two peaks of hydroxycinnamic acid were obtained (peaks 5 and 6). The cy peaks represented ca. 98% of the total area at 525 nm, while the pelargonidin peak (peak 3) was ca. 2%. The variation of the flavonoid content between the harvest of 2003 and 2004 was significant (p 0.05) for cy (256 and 138 mg/100 g f.w.), pelargonidin (3.6 and 2.7 mg/100 g f.w.), quercetin (14.1 and 15.3 mg/100 g f.w.) and hydroxycinnamic acid (12.4 and 14.1 mg/100 g f.w.), respectively. In spite of the difference between harvesting periods, the anthocyanin contents were much higher than those found by Gerasopoulos and Stavroulakis (1997), of 10 mg/100 g f.w. in Morus nigra cv. Mavrournia and 1 mg/100 g f.w. in Morus alba cv. Mavri cultivated in Greece.

0 (c)

7 400

0 0

5

10 15 20 25 Retention time (minutes)

30

35

Figure 1. HPLC chromatogram from wild mulberry at 270 nm. Peaks 1 and 2, cy derivates; peak 3, pelargonidin derivate; peak 4, quercetin derivate; peaks 5 and 6, hydroxycinnamic acids; peak 7, cy (A), methanol elute; (B), methanol : ammonia elute; (C) acidic hydrolyses of peaks 1 and 2.

Spectral Characterisation of Anthocyanins Two pigments were purified from methanol eluate after SPE by paper chromatography, corresponding to peaks 1 (Pigment 1) and 2 (Pigment 2), and their structural identification was performed by UV-V spectra and mass spectrometry (MS/MS). According to Harborne (1958, 1967) important structural information can be provided by spectral data, including the aglycon identity, sugar moiety position and presence of the cinnamoyl group. The anthocyanidins and their glycosylated forms, anthocyanins, exhibit broad absorption maximal in the visible region in acidic solution (465–550 nm) and have a less intense maximal at about 275 nm (Table 1).


Anthocyanins from Wild Mulberry (Morus Nigra L.)

21

major anthocyanins found in mulberry are not acylated. In addition, the alkaline hydrolysis of anthocyanin is another test that can indicate the presence or absence of the cinnamoyl group in the moiety, since saponification specifically removes quantitatively an acyl group leaving the sugars of anthocyanin intact. The results from saponification of mulberry anthocyanins confirmed that Pigments 1 and 2 did not contain the cinnamoyl group, since the wavelength and retention times of peaks 1 and 2 (Table 1) were not modified.

In acidified methanolic solution, the maximum wavelength ( max) of Pigments 1 and 2 were 531 and 529 nm, respectively. The acidic hydrolysis of both pigments resulted in one major anthocyanidin with max at 537 nm (Figure 1(C), similar to the value cited in literature for cy. The other common anthocyanidins show different max (malvidin, 542 nm; petunidin, 543; delphinidin, 546 nm; peonidin, 532; pelargonidin, 520 nm) (Harborne, 1958 and 1967). Moreover, the retention time and spectra of the hydrolysed pigment matched with those of cy. Spectral measurements are valuable in determining the position of sugar attachment in the anthocyanidin molecule. The two most common classes of anthocyanin, the 3- and 3,5-diglycosides, have similar spectra but show differences in intensity at the region of 410 to 450 nm. These differences have been traditionally expressed in terms of E440/E max ratio, where the 3-glycosides exhibiting ratios that are about twice as large as those for the 3,5-diglycosides and 5-glycosides (Harborne 1967). Then, the E440/E max ratio of 23% obtained for both pigments is an indicator of 3- or 3,5-glycosilation (Table 1). Moreover, the o-dihydroxy grouping at the B-ring was free since the mulberry pigments showed bathocromic shift in the presence of the aluminium ion. According to Harborne (1958), the confirmation of the presence (or absence) of aromatic acid as a cinnamoyl component can be made by spectral means. The ratio of absorbance at the cinnamoyl maximum (340 nm) to the absorbance at the anthocyanin maximum wavelength (520 nm), 340 (acyl)/ max, is a measure of the molar relation of the cinnamic acid to the anthocyanidin. In acidified methanolic solution, a ratio of 48% to 71% is indicative of a 1 : 1 molar ratio, while a ratio of 83% to 107% is characteristic of a 2/1 molar ratio of cinnamic acid to anthocyanidin. A ratio of less than 40% is an indicative of the absence of the cinnamoyl group. The 340 (acyl)/ max ratios found for the mulberry anthocyanins were 10% for both pigments (Table 1). These ratios suggest that the two

Carbohydrates Attached to Anthocyanins The carbohydrates obtained by acidic hydrolysis of pigments 1 and 2 were subjected to analysis by GC-EIMS, resulting in 4 peaks (Figure 2) and 2 peaks (Figure 3), respectively. The 4 peaks obtained from pigment 1 resulted in only two different mass spectra, one corresponding to glucose and the other to rhamnose. The two peaks obtained from pigment two gave the same mass spectra, corresponding to glucose. The presence of two peaks for the same sugar at the chromatogram was expected since all monosaccharides with five or more carbon atoms in the backbone occur as cyclic and straight-chain form derivates, which have different retention times. Anthocyanins Identification by MS ESI-MS/MS is a good tool for anthocyanin analysis because it does not require derivatisation and the positive charge of anthocyanidins at low pH values permits their easy detection using low voltages, since other potentially interfering compounds usually are not ionised (Giusti et al., 1999). After a final purification by paper chromatography, the two pigments obtained were analysed individually by MS/MS using direct injection into the spectrometer. For Pigment 1, a molecular ion [M] was found at m/z 595 (Figure 4(A))

Table 1. Spectral data of pigments isolated from Brazilian wild mulberry (Morus nigra L.). UV-V (CH3OH-HCl 0.1%) Anthocyanin Pigment (P)

max(nm)

Abs440/Emax (%)

AlCl3 max(nm)

AlCl3 ( )

P1 P1 (alkaline hydrolyse) P1 (acidic hydrolyse) P2 P2 (alkaline hydrolyse) P2 (acidic hydrolyse) Cy1,2 Cyanidin-3-glucoside1,2 Cyanidin-3,5-diglucoside1,2

281,531 281,531 277,537 282,529 281,529 279,537 277,535 274,523 273,524

23 25 22 23 26 22 19 24 13

574

43

575 574

38 45

574

38 18

1

Harborne, 1958. 2Harborne, 1967. Downloaded from http://fst.sagepub.com at UNIV DE SAO PAULO BIBLIOTECA on March 13, 2007 © 2007 SAGE Publications. All rights reserved. Not for commercial use or unauthorized distribution.

Abs340/Absmax(%) 13 23 10 20

20 20

22


and the fragmentation yielded two daughter ions at m/z 449 [cy glu], resulting from the loss of a rhamnose moiety, and the other at m/z 287 [cy] that results from the loss of a glucose residue (Figure 4(B)). According to the data obtained from UV-V characterisation and MS/MS spectra, it could be concluded that Pigment 1 corresponded to cyanidin-3-glucosylrhamnoside and that glucose is the sugar attached directly to the cy structure, since the fragment ion m/z 449 was detected. According to Garcia-Beneytez et al. (2003), the anthocyanin from grape peel with m/z at 595 was identified as cyanidin-3-O-p-cumaroilglucoside. In the case of mulberry, GC-MS analyses showed rhamnose and glucose as the two carbohydrates attached to the aglycon. Moreover, the Abs340/Abs max ratio of Pigment

1 was lower than 40%, which means a lack of the cynnamoyl group. Pigment 2 revealed a molecular ion [M] 449 (Figure 5(A)). MS2 of the molecular ion yielded one fragment at m/z 287 (Figure 5(B)) resulting from the loss of a glucose residue. Pigments with the same mass were previously detected by Dugo et al. (2001) from black bilberry and identified as cyanidin-3-glucoside, cyanidin-3-galactoside and petunidin-3-arabinoside. Considering the m/z 287 as being cyanidin and that the carbohydrate attached to Pigment 2 was identified by GC-MS as being glucose, plus the data from UV/visible characterisation, it can be concluded that the anthocyanin isolated from wild mulberry corresponds to cyanidin-3-glucoside.

a b 204 204 73

Abundance

73 191

b 147

147

191

217

217 50

100

150

200

250

a

50

100

150

m/z

0

10

200

250

300

m/z

20

30

40

50

60

Retention time (minutes)

Figure 2. GC-EI-MS chromatogram of carbohydrates released by acid hydrolysis of Pigment 1 isolated from wild mulberry. (a) rhamnose (mass spectra on the right); (b) glucose (mass spectra on the left).

a 204

Abundance

73 191

a 147

50

100

150

217

200

250

300

m/z

0

10

20

30

40

Retention time (minutes)

50

60

Figure 3. GC-EI-MS chromatogram of carbohydrates released by acid hydrolysis of Pigment 2 isolated from wild mulberry. (a) glucose (mass spectra on the right).


Anthocyanins from Wild Mulberry (Morus Nigra L.) 595.32

100

(a)

Percentage

MS

23

however, instead of three derivatives, the authors also reported the presence of cyanidin-3-sophoroside, pelargonidin-3-glucoside and pelargonidin-3-rutinoside. Anthocyanidin profile has been shown to vary largely among berries: while raspberry presented only cy and pelargonidin derivatives, similar to mulberry, the six most common anthocyanidins were found in cranberry (Wu and Prior, 2005). Antioxidant Activity

(b)

287.30

100

OH

m/z 287

Percentage

MS2

(595)

600

590

580

570

560

550

540

530

520

510

500

0

OH

O

HO

CONCLUSION m/z 449

O

m/z 595

glucose

rhamnose

OH 595.52 449.42

600

590

580

570

560

550

540

530

520

510

0 500

The CME, FN and FA from wild mulberry were tested for antioxidant activity by using the -carotene bleaching method (Figure 6). The CME showed antioxidant activities of 53 ± 2%, at 10 M GAE, and FN and FA showed 44 ± 2% and 50 ± 2%, respectively, at 1 M GAE. For comparison, at 10 M, the synthetic antioxidant BHT gave an inhibition of 34 ± 3%, the flavonol quercetin a 23 ± 3% inhibition and the anthocyanidins, cy and pelargonidin a 54 ± 4% inhibition and 53 ± 2% inhibition, respectively (Figure 6).

Figure 4. MS and MS/MS spectra of cyanidin-3-glucosylrhamnoside (Pigment 1) performed with an electron spray source in the positive mode (MS) and by argon collision (MS2) from m/z 595 parent ion from mulberry anthocyanin.

Among flavonoids present in mulberry, cy derivates were the major components, and pelargonidin and quercetin the minor ones. Two cy derivates were purified and characterised by UV-V spectra and MS/MS spectra and identified as cyanidin-3-glucoside and cyanidin-3-glucosylrhamnoside, responsible for 71 and 19%, respectively, of the total cy content. In spite of the mulberry studied here being a wild fruit, the anthocyanin content is higher than that reported for other cultivated mulberry and can be a potential source of anthocyanins in the diet.

ACKNOWLEDGEMENTS The composition of the anthocyanins identified in Brazilian wild mulberry was different from that reported for Morus nigra cv. Mavromournia, in which cyanidin-3-sophoroside (4.3%), cyanidin-3-glucorutinoside (63.5%), and pelargonidin-3-glucoside were identified (Gerasopoulos and Stravoulakis, 1997). Dugo et al. (2001), on the other hand, also identified cyanidin-3-glucoside and cyanidin-3-rutinoside as the main anthocyanins of a commercial Morus nigra cv.;

The authors would like to acknowledge Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Pesquisa e Desenvolvimento Cientifíco e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support and scholarship. We would also like to thank Laboratório de Biofarmacotécnica (BIOFAR-USP) for the facilities given for the realisation of part of the experimental work.


24


100

(a)

449.20

Percentage

MS

0 300

320

340

360

380

400

420

440

460

480

(b)

287.42

100

m/z 500

OH OH

o

HO

MS2 (449)

o

m/z 449 glucose

Percentage

OH

0 100

150

200

250

300

350

400

m/z 450

Figure 5. MS and MS/MS spectra of cyanidin-3-glucoside (Pigment 2) performed with an electron spray source in the positive mode (MS) and by argon collision (MS2) from m/z 449 parent ion from mulberry anthocyanin.

CME FN FA Cyn Pg Q BHT 0

10

20

30

40

Oxidation inhibition (%)

50

60

70

Figure 6. Antioxidant activities of CME at 10 M GAE, FN and FA at 1 M GAE, obtained from mulberry (Morus nigra L.) and assayed by the -carotene bleaching method. Controls were cy, pelargonidin, quercetin and BHT at 10 M.


Anthocyanins from Wild Mulberry (Morus Nigra L.)

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