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TRANSCRIPT
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: [email protected]
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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.
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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
1
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484
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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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