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

IOANNA ILIOPOULOU,ᵻ DELPHINE THAERON,‡ ASHLEY BAKER, ‡ ANITA JONES,ᵻ 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; E-mail: neil.robertson@ed.ac.uk

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

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

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

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

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

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

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

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

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

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

induced colour changes, under normal cooking conditions.

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

UV/Vis spectroscopy; CIE colour measurements

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INTRODUCTION

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

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

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

is also first judged from its colour 1.

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

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

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

impact during pasteurisation, sterilisation or concentration enhances the formation of

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

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

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

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

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

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

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

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

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

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

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

glucosidic molecules 7-9. Anthocyanins are responsible for the intensive red colour of black

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

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

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

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

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

the nature of the natural dyes 3.

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

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

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

moiety and formation of aglycon was suggested by Adams (1973) 16. This study also

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

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

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

products for anthocyanin 3,5 diglycosides 4,17.

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

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

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

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

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

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

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

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

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

Black carrot consists of a high ratio of mono-acylated anthocyanins 10-13. The question that

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

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

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

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

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

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

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

only the total anthocyanin concentration can be approximated.

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

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

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

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

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

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

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

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

enhance the stability of this commercially available natural colourant.

MATERIALS AND METHODS

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

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

was stored at -18 oC.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

vacuum on a freeze-drier.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

NMR to assign aromatic peaks. High-resolution 1H NMR spectra of the purified

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

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

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

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

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

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

collected using Spectra ManagerTM II Software.

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

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

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

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

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

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

produced by heat treatment was calculated using the ΔE* = [(ΔL*) 2 + (Δa*) 2 + (Δb*) 2] 1/2

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

RESULTS AND DISCUSSION

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

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

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

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

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

enable assignment of the aromatic protons.

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

anthocyanins can vary 11. Using RP-HPLC, five major anthocyanin components of black

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

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

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

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

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

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

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

Gläβgen’s study (1992) 28. The NMR results from the present study (Table 2) show that

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

appeared in the spectrum for compound 5.

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

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

determined and to be followed during thermal degradation. The well-resolved region

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

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

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

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

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

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

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

variation in the composition of the mixture during thermal degradation.

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

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

solid powder.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

commonly used UV/Vis data.

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

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

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

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

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

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

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

the dark (Figure S10).

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

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

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

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

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

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

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

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

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

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

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

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

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

above.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

degradation mechanism between solid and aqueous conditions.

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

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

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

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

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

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

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

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

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

mixture to be quantified.

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

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

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

was superior to that of the solutions.

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

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

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

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

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

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

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

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

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

REFERENCES

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

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

Campden BRI. 2011, Available from:

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

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

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

(3) Sadilova, E.; Reinhold, C.; Stintzing, C.F. Thermal degradation of anthocyanins and its

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

1471.

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

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

Sci. and Techn. 2010, 21, 3-11.

(5) Mullen, W.; Edwards, C.A.; Serafini, M.; Crozier, A. Bioavailability of pelargonidin-3-O-

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

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

(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.

(7) Türker, N.; Erdoğdu, F. Effects of pH and temperature of extraction medium on effective

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

Food Eng. 2006, 76, 579-583.

(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.

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

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

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

215.

(10) Elham, G.; Reza, H.; Jabbar, K.; Parisa, S.; Rashid, J. Isolation and structure

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

Pap. of Bio. Sci. 2006, 9, 2905- 2908.

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(11) Montilla, C. E.; Arzaba R. M.; Hillebrand, S.; Winterhalter, P. Anthocyanin composition

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

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

(12) Turker, N.; Aksay, S.; Instanbullu, O.; Artuvan, E. A study on the relation between

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

2007, 30, 953-969.

(13) Schwarz, M.; Wray, V.; Winterhalter, P. Isolation and identification of novel

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

2004, 52, 5095-5101.

(14) Hrazdina, G. Reactions of the anthocyanidin-3,5-diglucosides. Formation of 3,5-di-(O-

β- 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.

(16) Adams, J. B. Thermal degradation of anthocyanins with particular reference to the 3-

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

1973, 24, 747-762.

(17) Von Elbe, J. H.; & Schwartz, S.J. Colorants. In O. R. Fennema (Ed.), Food Chem. (3rd

ed.); Markel Dekker, Inc.: New York, 1996; pp. 651-722.

(18) Mazza, G.; Brouillard, R. Colour stability and structural transformations of cyanidin-3,5-

diglucoside and four 3-deoxyanthocyanins in aqueous solutions. J. Agric. Food Chem.

1987, 35, 422-426.

(19) Lapidot, T.; Harel, S.; Akiri, B.; Granit, R.; Kanner, J. pH-dependent forms of red wine

anthocyanins as antioxidants. J. Agric. Food Chem. 1999, 47, 67-70.

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(20) Março, H. P.; Scarminio, S. I. Q-mode curve resolution of UV-vis spectra for structural

transformation studies of anthocyanins in acidic solutions. Anal. Chim. Acta. 2007, 583,

138-146.

(21) Brouillard, R. Origin of the exceptional colour stability of the zebrina anthocyanin.

Phytochem. 1981, 20, 143-145.

(22) Brouillard, R. The in vivo expression of anthocyanin colour plants. Phytochem. 1983, 22,

1311-1323.

(23) Dangles, O.; Saito, N.; & Brouillard, R. Anthocyanin intramolecular copigment effect.

Phytochem. 1993, 34, 119-124.

(24) Francis, F. J.; Markakis, C.P. Food colourants: anthocyanins. Cri. Rev. in Food Sci.

Nutr. 1989, 28, 273- 314.

(25) Goto, T.; Kondo, T.; Tamura, H.; Imagawa, H. Structure of gentiondelphin. An acylated

anthocyanin isolated from gentiana makinoi, that is stable in dilute aqueous solutions.

Tetr. Letters. 1982, 23, 3695-3698.

(26) Resolution Oeno. Determination of chromatic characteristics according to CIElab.

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

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

(27) Rodriguez-Saona, E. L.; Wrolstad, E. R. Extraction, isolation, and purification of

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

[Published online: 1 August 2001]. 2001; Available from:

http://onlinelibrary.wiley.com/doi/10.1002/0471142913.faf0101s00/full

(28) Gläβgen, E. W.; Wray, V.; Strack, D.; Metzger, W. J.; Seitz, U. H. Anthocyanins from

cell suspension cultures of Daucus carota L. Phytochem. 1992, 31, 1593-1601.

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

Anthocyanins from wild carrot suspension cultures acylated with supplied carboxylic

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

(30) Bąkowska-Barczak, A. Acylated anthocyanins as stable, natural food colourants - a

review. Polish J. Food Nutr. Sci. 2005, 14/55, 107-116.

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

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

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

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

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

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

Enterprise/EPRSE industrial Case award (11330371). Open data:

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

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FIGURE CAPTIONS

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

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

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

coum-xy-glc-galactoside

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

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

signals

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

pH 6.8

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

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

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

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

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

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

the eye

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

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

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

the eye

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

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

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

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

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

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

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

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

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

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

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

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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+]

1

Cyanidin-3-xy-glc-

galactoside

15.4 743.2

2

Cy-3-xy- galactoside

16.3 581.2

3

Cy-3-sin-xy-glc-

galactoside

17.1 949.2

4

Cy-3-fer-xy-glc-

galactoside

17.6 919.2

5

Cy-3-coum-xy-glc-

galactoside

18.2 889.2

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Table 2: 1H-NMR Chemical Shifts of Aromatic Protons for the Anthocyanin Fractions in MeOD-d4-TFA-

d (see Table 1 for Proton Numbering Scheme).

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FIGURES

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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Figure 11

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65234

TOC Graphic

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