Analysis of the Thermal Degradation of the Individual Anthocyanin

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Analysis of the Thermal Degradation of the Individual Anthocyanin Components of Black Carrot (Dausus carota L.) – A New Approach Using High-Resolution 1H NMR Spectroscopy Ioanna Iliopoulou, Delphine Thaeron, Ashley Baker, Anita C Jones, and Neil Robertson J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02543 • Publication Date (Web): 10 Jul 2015 Downloaded from http://pubs.acs.org on July 12, 2015

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Journal of Agricultural and Food Chemistry

Analysis of the Thermal Degradation of the Individual Anthocyanin Components of Black Carrot (Daucus carota L.) – A New Approach Using High-Resolution 1H NMR Spectroscopy ᵻ

ᵻ

IOANNA ILIOPOULOU, DELPHINE THAERON,‡ ASHLEY BAKER, ‡ ANITA JONES, NEIL ROBERTSON* ᵻ

ᵻ

EaStCHEM School of Chemistry, Joseph Black Building, David Brewster Road, Edinburgh, United Kingdom EH9 3FJ, ‡

Macphie of Glenbervie, Stonehaven, United Kingdom, AB39 3YG

*Author to whom correspondence should be addressed. Telephone +44 131 6504755; Email: [email protected]

1

ACS Paragon Plus Environment


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The black carrot dye is a mixture of cyanidin molecules, the NMR spectrum of which shows a

2

highly overlapped aromatic region. In this study, the 1H NMR (800 MHz) aromatic chemical

3

shifts of the mixture were fully assigned by overlaying them with the characterised 1H NMR

4

chemical shifts of the separated components. The latter were isolated using RP-HPLC and

5

their chemical shifts were identified using 1H and 2D COSY NMR spectroscopy. The stability

6

of the black carrot mixture to heat exposure was investigated at pH 3.6, 6.8 and 8.0 by heat-

7

treating aqueous solutions at 100 oC and the powdered material at 180 oC. By integrating

8

high-resolution 1H NMR spectra it was possible to follow the relative degradation of each

9

component, offering advantages over the commonly used UV/Vis and HPLC approaches.

10

UV/Vis spectroscopy and CIE colour measurements were used to determine thermally

11

induced colour changes, under normal cooking conditions.

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KEYWORDS: Anthocyanins; Cyanidin, Thermal degradation; NMR Integration; acylation;

13

UV/Vis spectroscopy; CIE colour measurements

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INTRODUCTION

18

The colour of a food or beverage is of paramount importance, as it is the first characteristic to

19

be noticed and one of the main ways of visually assessing the food before consuming it. The

20

perceived colour provides an indication of the expected taste of food and the quality of a food

21

is also first judged from its colour 1.

22

Many raw foods, such as fruits and vegetables, have vibrant, attractive colours. However,

23

upon processing, their colour may fade or be completely lost. Most natural colours are highly

24

labile towards temperature, pH, oxygen and light during processing and storage. The thermal

25

impact during pasteurisation, sterilisation or concentration enhances the formation of

26

degradation products and the concomitant colour loss. Consequently, the food products may

27

cease to be attractive to consumers 2. Thus, it is important to understand the conditions

28

governing colourant degradation in order to establish measures to avoid its occurrence 3.

29

Research over the past decades has produced incontrovertible evidence of the health benefits

30

arising from the consumption of many fruits and vegetables. Many researchers have tried to

31

identify the health- promoting ingredients of flavonoids, a class of phenolic. Most prominent

32

amongst the flavonoids are the anthocyanins, one of the most abundant constituents

33

responsible for the attractive red, blue and purple colours in many fruits and vegetables. They

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are widely found in berries, dark grapes, cabbages, red wine, cereal grains and flowers 4-6.

35

Anthocyanins are derivatives of salts called anthocyanidins 7; they occur in nature as

36

glycosides of anthocyanidins and may have aliphatic or aromatic acids attached to the

37

glucosidic molecules

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carrot. The anthocyanin profile of black carrot has been analysed in the past and found to

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consist mainly of cyanidin-based dyes 10-13 (Table 1).

7-9

. Anthocyanins are responsible for the intensive red colour of black

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Thermal treatment can result in pigment breakdown and/or a variety of degradation species,

41

depending on the nature of the anthocyanins and the severity of the heat-treatment 4. Sadilova

42

et al. (2007) identified, by HPLC, different thermal degradation compounds which depend on

43

the nature of the natural dyes 3.

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Previous studies suggest possible mechanisms for the degradation of anthocyanins. Amongst

45

them, opening of the pyrylium ring and formation of chalcone as an initial step was proposed

46

by Hrazdina (1971) and Markakis (1957) 14-15. On the other hand, hydrolysis of the glycosidic

47

moiety and formation of aglycon was suggested by Adams (1973)

48

confirmed that anthocyanins are degraded during heating into a chalcone structure which in a

49

second step involves transformation into a coumarin glycoside with a B-ring loss. Von Elbe

50

and Schwartz (1996) also suggested that coumarin 3,5-diglycosides are common degradation

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products for anthocyanin 3,5 diglycosides 4,17.

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During heat exposure, the stability of anthocyanins depends on the composition and the

53

characteristics of the medium, with pH playing an important role. Anthocyanins adopt

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different chemical structures which exist in pH-dependent equilibrium 18-20.

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Some studies indicate that acylated anthocyanins, mainly those with planar aromatic

56

substituents, exhibit greater stability, especially when kept in aqueous solutions, and play an

57

important role in increasing the thermal stability of the dye compared to the non-acylated

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counterparts. It is believed that the aromatic residues of the acyl groups stack with the

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pyrylium ring of the flavylium cation which reduces the likelihood of the hydration reaction

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in the vulnerable C-2 and C-4 positions 21-25.

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Black carrot consists of a high ratio of mono-acylated anthocyanins

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arises is; are black carrot acylated anthocyanins more stable compared to the non-acylated

16

. This study also

10-13

. The question that

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ones? In other words, does the structure of anthocyanins affect the stability?

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Previous studies of black carrot have typically used HPLC and UV/Vis analysis for

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quantification of the components, but there are some weaknesses to these techniques. For

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HPLC, there are concerns about pH-dependent variation in the wavelengths of absorption

67

maxima and the values of absorption coefficients, leading to unreliable quantification. Using

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UV/Vis spectroscopy alone, it is impossible to resolve and quantify the separate components;

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only the total anthocyanin concentration can be approximated.

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In the present study, 1H NMR spectroscopy and signal integration was used to investigate the

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thermal degradation of the individual anthocyanin components of a commercial black carrot

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concentrate, and the effect of pH on this degradation. Complementary UV/Vis spectroscopy

73

and CIE colour space measurements 26 were used to follow colour degradation. Separation of

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the mixture into individual components, assignment of each component followed by

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integration of 1H NMR signals in spectra of the mixtures were the steps used. The resulting

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understanding of the relative stabilities of the different components, in particular the role of

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structure (acylation) on the stability should be valuable in developing future strategies to

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enhance the stability of this commercially available natural colourant.

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80

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MATERIALS AND METHODS

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Plant Materials. Commercial concentrate of black carrot (Daucus carota L.) was supplied

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by Naturex Ltd (manufacturer’s code: COPG4167, sample code: G00017). The concentrate

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was stored at -18 oC.

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Solvents and Reagents. Deuterated NMR solvents were purchased as follows: methanol-d4

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from Sigma Aldrich (USA) trifluoroacetic acid-d from Sigma Aldrich (USA) or Cambridge

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Isotope Laboratories (CIL) (USA). Hydrochloric acid S.G. 1.18 (≈ 37%), sodium hydroxide

88

(97%) and sodium dihydrogen orthophosphate dihydrate were purchased from Fisher

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Scientific (UK). Disodium hydrogen phosphate dihydrate and citric acid monohydrate were

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obtained from Sigma Aldrich (USA). Acetonitrile and water for HPLC were purchased from

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VWR International. All the HPLC solvents were of analytical grade. C-18 Cartridges Vac.

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35cc (10 g) (WAT043345)) purchased from Waters (Ireland, U.K).

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Sample Preparation. A two-step extraction process was applied to the black carrot sample to

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remove non-anthocyanin components. 100 g of black carrot concentrate was mixed with 150

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mL of chloroform in a separating funnel and left overnight. The aqueous phase was collected

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and further purified by solid-phase extraction 27, using mini columns (C-18 Cartridges Vac.

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35cc (10 g) (WAT043345)) purchased from Waters (Ireland). The eluent of the extraction

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(methanolic mobile phase), was concentrated in a rotary evaporator (IKA® RV 10 basic) at

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25 oC and further dried under vacuum, using liquid nitrogen, yielding 5 g of powder.

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High Performance Liquid Chromatography. 100 mg of the extracted black carrot powder

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(see section 2.3) were dissolved in 1 mL of distilled water. Semi-preparative reverse-phase

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high-performance liquid chromatography (RP–HPLC) was performed on an HP1100 system

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equipped with a semi-preparative C18 Agilent column Eclipse XDB-C18 (9.4x250mm i.d., 5

104

µm) at a constant temperature of 20 oC and a flow rate of 2 mL/min. A mobile phase gradient

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was used for elution; eluent A consisted of water with 0.1 mL formic acid and eluent B of

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acetonitrile (ACN) and water with 0.1% formic acid (1:1). The elution profile was 10% B at

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0min, 35% of B at 10min, 50% of B at 35min, 80% of B at 40min and 10% of B at 45min.

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The injection volume was 20 µL and the detector was set at 520 nm. The fractions were

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transferred into vials and mass spectrometric analysis performed on an Agilent Series 1100 6


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HPLC system fitted with an electrospray ionization (ESI) source. Repeated injections were

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performed, and the isolated fractions were combined until a mass of 2-5 mg per fraction was

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obtained. The purified fractions were frozen using dry ice and acetone, and then dried under

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vacuum on a freeze-drier.

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Heating Experiments. A domestic oven (Dēlonghi E012001W) was used to heat-treat

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aqueous solutions with pH values of 3.6, 6.8 and 8.0. Citric acid/ phosphate and phosphate

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buffers were used to adjust the pH. Hydrochloric acid and sodium hydroxide were also used

117

where necessary for adjusting the pH, to avoid salts which interfere with the HPLC column.

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The samples were heated in an oven at around 180 oC, to maintain the aqueous sample

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temperature at 100 oC, for periods up to 100 minutes. A 60-minute heat-treatment was also

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applied to samples for which the pH ranged between 3.4 and 8.2.

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Powder samples of black carrot were prepared by dissolving black carrot in aqueous

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solutions, adjusting the pH to 3.6 and to 6.8, followed by freeze-drying. The powders were

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then exposed to heat in a high performance furnace (CARBOLITE® (UK), at 180 oC).

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NMR Spectroscopy. A mixed solvent consisting of 10 g MeOH-d4:0.5 mL trifluoacetic

125

acid-d (TFA-d) was used for all NMR measurements. The structures of the compounds

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isolated by RP-HPLC were determined using 1D 1H-NMR analysis on a Bruker 500 MHz

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spectrometer (10 mg in 0.8 mL MeOH/TFA), in combination with two-dimensional COSY

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NMR to assign aromatic peaks. High-resolution

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(unseparated) black carrot (10 mg in 0.8 mL of MeOH/TFA) were acquired on a Bruker 800

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MHz spectrometer. In addition, 1H NMR spectra (800 MHz) used for integration of the

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samples exposed to heat were also acquired (10 mg in 0.8 mL of MeOH/TFA).

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UV/Vis Spectroscopy. Absorption spectra in the visible region (300-800 nm) were recorded

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using a Jasco V-670 series spectrophotometer. Solutions (40 µL of sample solution in 3 mL

1

H NMR spectra of the purified

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citric acid/phosphate buffer) were contained in a quartz cell (d = 1 cm) and the data were

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collected using Spectra ManagerTM II Software.

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Colour Measurements. The Spectra ManagerTM II Software was used to calculate the CIE

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lab coordinates using the Jasco V-670 series spectrophotometer (Tokyo, Japan). Chroma

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value C*[C* = a*2 + b*2)1/2] and hue angle ho [ho = arctan (b*/a*)] were calculated from

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parameters a* (from green to red) and b* (from blue to yellow) values. The hue angles were

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expressed on a 360o colour wheel, in which 0 and 360o represent red, 90o yellow, 180o green

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and 270o blue. The illuminant was D65 and the observer angle was 10o. The change in colour

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produced by heat treatment was calculated using the ∆E* = [(∆L*) 2 + (∆a*) 2 + (∆b*) 2]

143

equation at pH 3.6, 6.8 and 8 for several time intervals 26.

1/2

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RESULTS AND DISCUSSION

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Isolation and Structure Characterisation – NMR Studies. As shown in Figure 1, even

147

after purification by solid-phase extraction, the NMR spectrum of the black carrot mixture

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contains many overlapping peaks, preventing the assignment of individual components. The

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extraction process has simplified the aromatic region (6.2 to 9 ppm) as shown by comparison

150

with Figures S1 and S2, but further information on the individual components is needed to

151

enable assignment of the aromatic protons.

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Montilla (2011) describes that for different black carrot species the composition of

153

anthocyanins can vary

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carrot were isolated, as shown in the chromatogram in Figure 2. Mass spectrometric analysis

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indicated that each fraction corresponds to a particular anthocyanin molecule (identifiable as

11

. Using RP-HPLC, five major anthocyanin components of black

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the molecular ion), as summarised in Table 1 (Figures S3-S7). These results are consistent

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with previous studies of the composition of black carrot 10-13.

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1D 1H-NMR and 2D COSY NMR analysis (Figures S3- S7) enabled assignment of each

159

aromatic proton of each compound (Tables 1 and 2). The chemical shifts are consistent with

160

the ones assigned for the anthocyanins from cell suspension culture of Daucus carota L. in

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Gläβgen’s study (1992)

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compounds 1 and 2 are the non-acylated components and compounds 3, 4 and 5 are the

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acylated anthocyanin compounds, confirming results from previous studies. It can also be

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seen that the presence of sinapic, ferulic and coumaric acids on the glucose moiety has an

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effect on the chemical shift of the cyanidin protons. For example, the chemical shifts of H-4

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in the non-acylated compounds are in the range δ 9.016 -9.010 while those for the acylated

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compounds appear at δ close to 8.540. The same effect is seen on the chemical shifts of the

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H-6 and H-8 protons. In Gläβgen’s study (1992) a low-frequency shift of H-4 protons of

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black carrot anthocyanins acylated with sinapic, ferulic and coumaric acids compared to the

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non-acylated counterparts was also noted 28. Also, Dougall (1998) described a marked effect

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of cinnamic or benzoic acids on the chemical shifts of the cyanidin H-4, H-6 and H-8 protons

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in the acylated compounds providing evidence for NMR shifting caused by acylation 29.

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The aromatic region of the black carrot mixture could be assigned completely with reference

174

to the NMR spectra of the five separated components. Each signal in the spectrum of the

175

mixture can be identified with a single signal or with overlapping signals from the spectra of

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the five compounds, as illustrated in Figure 3. In the region between δ = 6.0 and 7.5 ppm

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there is considerable overlap between peaks of the individual components in the spectrum of

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the mixture. On the other hand, in the region between δ = 7.8 and 9 ppm the individual

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component peaks are generally well resolved. The small intensity doublets in the region δ =

28

. The NMR results from the present study (Table 2) show that

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7.55, highlighted in red, and δ = 6.25 (overlapped), are attributed to minor impurities and also

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appeared in the spectrum for compound 5.

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Thermal Degradation Studies. The clear assignment of the NMR peaks in the black carrot

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mixture enables the fractional concentration of compounds 1 – 5 to be unambiguously

184

determined and to be followed during thermal degradation.

185

between δ = 8.4 – 9 ppm was used for integration to determine the composition of the

186

mixture and to quantify the degradation of each component. Specifically, the integrals were

187

determined for the combined H-4 proton signals of components 1 and 2 at δ = 9 ppm and the

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individual H-4 proton signals of the other three components in the region around 8.5 ppm.

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Overall, the expected general trend was noticed; the longer the exposure to heat, the more the

190

integrated NMR signals of the compounds were reduced. However, it was also apparent that

191

the integrals of the individual components were decreasing at different rates, resulting in

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variation in the composition of the mixture during thermal degradation.

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Before examining the NMR results in detail, we first describe the general degradation

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behaviour observed in the UV/Vis spectra of the anthocyanin mixture in solution and as a

195

solid powder.

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UV/Vis Spectroscopy and Colour Measurements. Black Carrot in Solution. Exposure to

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heat at pH 3.6 for 100 minutes resulted in an increase in lightness, L*, by 7.99 units,

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insignificant change in the hue angle ho and a decrease in the chroma, C*, by 16.13 units

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(Table S1). This indicated that the colour of the anthocyanins was fading with slight change

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of the hue. The UV-Visible spectra (Figure 4a) showed a decrease in absorbance but the λmax

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was not notably shifted. Heat-treatment at pH 6.8 for 100 minutes also resulted in an increase

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in lightness (8.65 units) and decrease in chroma (22.26 units), but there was also an increase

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in the hue angle by 18.12 units (Table S1). Therefore, in neutral conditions, the colour not

The well-resolved region

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only faded but the hue also changed from red to a more orange shade. The UV-visible spectra

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(Figure 4b) showed both a decrease in absorbance and a slight bathochromic shift in the λmax.

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To explore the pH-dependence of these effects, the UV/Vis spectra of the black carrot heat-

207

treated for 60 minutes at a range of pH values, from 3.4 to 8.2, were recorded. As shown in

208

Figure 5, with increasing pH, the λmax shifted bathochromically and the absorbance maximum

209

was noticeably decreased. Observing the CIE lab parameters; the lightness gradually

210

enhanced by 25.89 units, the ho increased dramatically to 70.43 units and the colour of neutral

211

and more basic solutions decolourised and gradually turned brown. The colour saturation

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decreased 40.52 units (Table S2). It is clear from the UV/Vis spectra that there is more rapid

213

degradation of the anthocyanins at higher pH values. This can be related to the different

214

forms of the anthocyanins present as a function of pH; in the case of the acidic solution, more

215

of the stable flavilium cationic form of anthocyanin is present, whereas in neutral conditions

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the percentage of less stable chalcone, carbinol and quinonoidal form will be increased.

217

Black Carrot Powders. To assess the effect of pH on solid-state samples, the pH was

218

adjusted to 3.6 and 6.8 before freeze-drying. After 60 min of heat-treatment, the colours

219

showed a slight increase in lightness (L*) by 2.73 and 5.03 units, respectively. The hue value

220

change was negligible for both cases and the chroma (C*) value decreased by 7.92 and 12.47

221

units, respectively (Table S1). The difference in the absolute absorbance between the two

222

samples is also negligible in the λmax of the UV-Vis spectra though (Figure 6 (a) and (b)). The

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UV-Vis spectra show that, at pH 3.6, the thermal stabilities of the solution and powder

224

samples are similar; however, at pH 6.8 the powder shows much higher stability than the

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solution. Comparing the two powder samples, it is evident that the pH prior to freeze-drying

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has little effect on the stability.

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NMR spectroscopy. Integration of the H-4 proton peaks in the aromatic region of NMR

228

spectrum (δ = 8.4 – 9), as indicated in Figure 3 enabled the percentage of each component in

229

the anthocyanin mixture to be quantified as a function of heating time, over a range of pH.

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The results are presented in Figures 7 to 11. As shown in the insets of the Figures, the overall

231

degradation of the total anthocyanin content determined from the NMR integrals follows a

232

similar trend to that derived from the UV/Vis absorbance. Notably, however, the absorbance

233

measurements imply a lesser extent of degradation than that quantified by the NMR data.

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This can be attributed to residual absorbance, in this spectral region, by the decomposition

235

products, which persists after degradation of the primary anthocyanin components. Thus, the

236

NMR data are able to give a better quantitative measurement of degradation than the

237

commonly used UV/Vis data.

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Black Carrot in Solution. As shown in Figure 7, at pH 3.6 all components showed

239

substantial degradation after heating for 100 minutes, but there was not complete destruction;

240

the total anthocyanin concentration was decreased by about 60%. In contrast, at pH 6.8 there

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was almost complete thermal degradation after 100 min with only about 10% of the total

242

anthocyanin content remaining. These observations are consistent with the expectation that

243

the anthocyanins are more stable in acidic conditions 18. In basic conditions (pH 8) we found

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complete degradation of black carrot solution after 48 hours storage at room temperature in

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the dark (Figure S10).

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The rate of degradation of the anthocyanin components can be assessed by considering the

247

percentage decrease in concentration after 60 min heating. At pH 3.6, the non-acylated

248

compounds, 1+2, showed the greatest rate of decomposition, resulting in a 43% decrease in

249

concentration in 60 minutes, compared with a decrease of about 30% for each of the acylated

250

components (Figure 7). At pH 6.8, the rate of decomposition of all the components was 12


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accelerated. However, the non-acylated compounds (1+2) decomposed more slowly than the

252

acylated compounds; the former showed a 66% drop in concentration after 60 minutes,

253

compared with a 75% drop for the latter (Figure 8).

254

The effect of pH on the composition of the thermally degraded anthocyanin mixture after

255

heating for 1 hour is illustrated in Figure 9. It is evident that the stability of all components

256

decreases significantly with increasing pH in the range pH 3.4 to pH 5.8. There is little pH-

257

dependence between pH 5.8 and 6.4 (a slight increase in stability is seen). An abrupt drop-off

258

in stability occurs as neutral pH is approached, with complete decomposition at pH 7 and

259

above.

260

To summarise, the thermal stability of all the anthocyanin compounds is greater under acidic

261

conditions, but the relative stability of acylated and non-acylated components is pH-

262

dependent. At low pH the acylated compounds are more stable than the non-acylated

263

compounds, but become less stable at higher pH. This is contrary to the general consensus in

264

the literature that acylation enhances the stability of anthocyanins at higher pH by protecting

265

the flavilium cation from nucleophilic attack by water molecules at C-2 and C-3 positions 21-

266

24, 30

267

of anthocyanin pigments with different degrees of acylation, rather than direct quantitation of

268

individual acylated and non-acylated components 21-24, 30.

269

Black carrot powders. To investigate the thermal degradation of black carrot in the solid

270

state, powder samples were prepared by freeze drying solution samples of pH 3.6 and 6.8.

271

After heating, the solid samples were dissolved and NMR spectra recorded.

272

The thermal degradation of powder and solution samples, after 1 hour’s heating, are

273

compared in Figures 10 and 11. (Note that the solids were effectively heated at a higher

. However, previous studies have been based on the comparison of the colour-stabilities

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temperature since no evaporation was occurring). For the samples at pH 3.6 (Figure 10) the

275

degradation properties of the powder are similar to those of the solution. At pH 6.8, there is

276

only a small overall increase in degradation in the powder compared to pH 3.6. In solution at

277

pH 6.8 however, the powder is significantly more stable than in solution. Regarding the

278

individual anthocyanins, it appears that changing the pH from acidic to neutral prior to

279

freeze-drying has little effect on the relative stability of the acylated and non-acylated

280

compounds in the resulting solid sample. This is consistent with the UV/Vis results (vide

281

supra). It is also notable that, at pH 6.8, the stability of the acylated components is markedly

282

higher in the powder than in the solution. These observations imply differences in the

283

degradation mechanism between solid and aqueous conditions.

284

To conclude, the anthocyanins components in a black carrot concentrate were successfully

285

isolated using HPLC and their chemical shifts were fully assigned using 1D 1H NMR and 2D

286

COSY NMR. The UV/Vis and the CIElab colour measurements showed an increased

287

degradation with increasing the pH and heating time. NMR measurements confirmed these

288

general trends. The summed NMR integrals of the five anthocyanin compounds followed a

289

similar trend to the UV/Vis absorbance. However, the UV/Vis data underestimate the extent

290

of degradation, as a result of the residual absorbance of decomposition species, making the

291

NMR method more accurate. Furthermore, integration of the H-4 proton peaks between the

292

region δ = 8.4 – 9, enabled percentage degradation of the individual components of the

293

mixture to be quantified.

294

The NMR results show that in acidic aqueous solution, there is enhanced stability of the

295

monoacylated compounds whereas in neutral conditions their stability is lower compared to

296

the non-acylated compounds. The thermal stability of powder samples, produced by freeze

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drying solutions at pH 3.6 and 6.8 was similar and the heat stability of the powders at pH 6.8

298

was superior to that of the solutions.

299

It is important to emphasise the benefits of NMR for studying individual anthocyanin

300

compounds, avoiding factors such as the pH dependence of the absorption, interfering

301

absorbing components and only-approximate knowledge of molar absorption coefficients at

302

different pH values, which are severe disadvantages using the common methods of HPLC

303

and UV/Vis. Although there are some limitations such as overlapping chemical shifts for the

304

compounds 1 and 2, reliable information can be gleaned on the relative stability of the

305

different anthocyanins. The observation of an unexpected effect of pH on the relative stability

306

of monoacylated and non-acylated anthocyanins emphasises the utility of NMR in providing

307

insight into the degradation of multi-component natural colorants, such as black carrot.

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309

310

311

312

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REFERENCES

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(1) Chapman, S. Guidelines on approaches to the replacement of tartazine, allura red,

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ponceau 4R, quinoline yellow, sunset yellow and carmoisine in food and beverages.

316

Campden

317

http://www.food.gov.uk/news/newsarchive/2011/sep/colourguidance

BRI.

2011,

Available

from:

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318 319

(2) Jackman, R.; Smith, J. Anthocyanins and betalains. In Nat. Food Col., eds. G. A.

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Hendry and J. D. Houghton, Springer: U.S., 1996; pp. 244-309

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impact on colour and in vitro antioxidant capacity. Mol. Nutr. Food Res. 2007, 51, 1461-

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(4) Patras, A.; Brunton, N, P.; O’ Donnell, C.; Tiwari, B. K. Effect of thermal processing on

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anthocyanin stability in foods; mechanisms and kinetics of degradation. Trends in Food

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glucoside and its metabolites in human following the ingestion of strawberries with and

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without cream. J. Agric. Food Chem. 2008, 56, 713-719.

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(6) Qin, C. G.; Li, Y.; Niu, W.; Shang, X.; Xu, C. Composition analysis and structural identification of anthocyanins in fruit of waxberry. Czech J. Food Sci. 2011, 29, 171-180.

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diffusion coefficient of anthocyanidin pigments of black carrot (Daucus carota var. L.). J.

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(8) Guisti, M.M.; & Wrolstad, R.E. Acylated anthocyanins from edible sources and their applications in food systems. Biochem. Eng. J. 2003, 14, 217-225.

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(9) Zozio, S.; Pallet, D.; Dornier, M. Evaluation of anthocyanin stability during storage of a

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coloured drink made from extracts of the andean blackberry (Rubus glaucus Benth.), acai

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(Euterpe oleracea Mart.) and black carrot (Daucus Carota L.). Fruits. 2011, 66, 203-

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characterization of anthocyanin pigments in black carrot (Daucus carota L.). Pakistan

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of black carrot (Daucus carota ssp. Sativus var. atrorubens Alef.). Cultivars antonina,

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beta sweet, deep purple, and purple haze. J. Agric. Food Chem. 2011, 59, 3385-3390.

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anthocyanin content and product quality: salgam as a model beverage. J. Food Qual.

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2007, 30, 953-969.

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pyranoanthocyanins from black carrot (Dausus carota L.) juice. J. Agri. Food Chem.

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2004, 52, 5095-5101.

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(14) Hrazdina, G. Reactions of the anthocyanidin-3,5-diglucosides. Formation of 3,5-di-(Oβ- D-glucosyl)-7-hydroxy coumarin. Phytochem. 1971, 10, 1125-1130. (15) Markakis, P.; Livingston, G. E.; Fellers, C. R. Quantitative aspects of strawberry pigment degradation. Food Res. 1957, 22, 117-30.

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glycosides of cyanidin. I. In acidified aqueous solution at 100 oC. J. Sci. Food Agri.

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chromatic characteristics. Compendium of international analysis of methods – OIV.

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[online]. 2006; Available: OIV-MA-AS2-11.pdf [accessed January 2006].

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anthocyanins. Current Protocols in Food Anal. Chem. Wiley Online Library.

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online:

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(29) Dougall, K. D.; Baker, C.D.; Gakh, G. E.; Redus, A. M.; Whittemore, A.N.

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Anthocyanins from wild carrot suspension cultures acylated with supplied carboxylic

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acids. Carbohydr. Res. 1998, 310, 177-189.

392 393

(30) Bąkowska-Barczak, A. Acylated anthocyanins as stable, natural food colourants - a review. Polish J. Food Nutr. Sci. 2005, 14/55, 107-116.

394 395

Supporting Information Available: 1H NMR spectra of the unpurified and partly

396

purified black carrot mixture; 1D, 2D COSY 1H NMRs and MS of the separated

397

compounds; tables of the CIE lab Colour measurements; description of the integral

398

quantification; 1H NMR spectra used for integration; HPLC data. This material is

399

available free of charge via the Internet at http://pubs.acs.org

400 401

We thank the Engineering and Physical Sciences Research Council for a Scottish

402

Enterprise/EPRSE

403

http://dx.doi.org/10.7488/ds/279.

industrial

Case

award

(11330371).

Open

data:

404 405 406 407 408 409 410 411 412 19



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413 414 415

FIGURE CAPTIONS

416 417

Figure 1: 1H NMR spectrum (800MHz) of black carrot mixture following purification by solid-phase extraction

418

Figure 2: HPLC profile of anthocyanin components of the specific black carrot species – 1: Cy-3-xy-glc-

419

galactoside, 2: cy-3-xygalactoside, 3: Cy-3-sin-xy-glc-galactoside, 4: Cy-3-fer-xy-glc-galactoside, 5: Cy-3-

420

coum-xy-glc-galactoside

421

Figure 3: NMR spectra of black carrot mixture (bottom, black) and the HPLC compounds 1 to 5.

422

* The singlet at δ = 8.08 ppm of Compound 2 and δ = 7.9 ppm of Compounds 3, 4 and 5 are solvent residual

423

signals

424

Figure 4: UV/Vis spectra as a function of time for heat-treatment of black carrot solution at (a) pH 3.6 and (b)

425

pH 6.8

426

Figure 5: UV/Vis spectra as a function of pH for heat treatment of black carrot solution for 60 min

427

Figure 6: UV/Vis spectra as a function of time for heat-treatment of black carrot powder, prepared by freeze-

428

drying of solution at (a) pH 3.6 and (b) pH 6.8

429

Figure 7: The percentage of each component (determined from NMR integrals) in black carrot solution as a

430

function of heating time, at pH 3.6. The inset compares the heating-time-dependence of the total anthocyanin

431

content derived from NMR data (green) with that derived from UV/Vis data (purple). The lines are a guide to

432

the eye

433

Figure 8: The percentage of each component (determined from NMR integrals) in black carrot solution as a

434

function of heating time, at pH 6.8. The inset compares the heating-time-dependence of the total anthocyanin

435

content derived from NMR data (green) with that derived from UV/Vis data (purple). The lines are a guide to

436

the eye

20


Page 21 of 35


437

Figure 9: The percentage of each component (determined from NMR integrals) in black carrot solution heated

438

for 60 minutes, as a function of pH. The inset compares the pH-dependence of the total anthocyanin content

439

derived from NMR data (green) with that derived from UV/Vis data (purple). The lines are a guide to the eye

440

Figure 10: Comparison of the effect of heating on a solution sample (red) at pH 3.6 and a powder sample (blue)

441

freeze-dried at pH 3.6. The percentage of each component (determined from NMR integrals) in each sample

442

after one hour’s heating is shown. (The solution sample was heated in a domestic oven at around 180 oC and the

443

solid sample was accurately heated in a furnace at 180 oC)

444

Figure 11: Comparison of the effect of heating on a solution sample (red) at pH 6.8 and a powder sample (blue)

445

freeze-dried at pH 6.8. The percentage of each component (determined from NMR integrals) in each sample

446

after one hour’s heating is shown. (The solution sample was heated in a domestic oven at around 180 oC and the

447

solid sample was accurately heated in a furnace at 180 oC)

21



Page 22 of 35

TABLES

Table 1: Molecular Structures of the Cyanidin Compounds which Comprise the Black Carrot Anthocyanin Mixture and Proton Numbering Schemes used in the Assignment of NMR Spectra Name

Anthocyanin Structure

Sugar Structure

Retention Time (min)

m/z [M+]

15.4

743.2

16.3

581.2

17.1

949.2

17.6

919.2

18.2

889.2

1 Cyanidin-3xy-glcgalactoside 2 Cy-3-xygalactoside 3 Cy-3-sinxy-glcgalactoside 4 Cy-3-fer-xyglcgalactoside 5 Cy-3-coumxy-glcgalactoside

1


Page 23 of 35


Table 2: 1H-NMR Chemical Shifts of Aromatic Protons for the Anthocyanin Fractions in MeOD-d4-TFAd (see Table 1 for Proton Numbering Scheme).

2



Page 24 of 35

FIGURES

Figure 1

3


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Figure 2

4



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Figure 3

5


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Figure 4

6



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Figure 5

7


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Figure 6

8



Page 30 of 35

Figure 7

9


Page 31 of 35


Figure 8

10



Page 32 of 35

Figure 9

11


Page 33 of 35


Figure 10

12



Page 34 of 35

Figure 11

13


Page 35 of 35


TOC Graphic OH

R"="Various"Sugars" " """"""""""""""pH" """"""""""""Heat"

O+

HO

OH R OH

NMR"

14


Analysis of the Thermal Degradation of the Individual Anthocyanin

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