Kinetic study of the equilibration between carminic acid and its two isomers isolated from cochineal dye

Article abstract of DOI:10.1248/cpb.c16-00838

Carminic acid (CA) is a major component of cochineal dye used in food additives, cosmetics, and pharmaceuticals. CA and its isomers, 2-C-α-glucofuranoside and 2-C-β-glucofuranoside of kermesic acid (DCIV and DCVII, respectively), were isolated from cochineal dye and the equilibrium constants (K) between CA, DCIV and DCVII were investigated. DCIV was partially converted to CA and DCVII, and DCVII was converted to CA and DCIV, whereas CA was very stable and only very slightly converted to DCIV and DCVII. Most of the DCIV and DCVII was converted to CA under aqueous conditions. The kinetic rate constants (k) for the degradation of DCIV within the first day of incubation at 24°C was determined to be 0.901 d^-1 and for the degradation of DCVII it was determined to be 1.102 d^-1. The k value for the formation of CA from the remaining DCIV was calculated to be 0.146 d^-1 and for the formation of CA from the produced DCVII it was found to be 0.148 d^-1. The K values were calculated as 1.22×10^-7, 2.61×10^-3 and 2.36×10^-3 mol/L for CA, DCIV and DCVII, respectively. These findings will be helpful for ensuring the safety and for aiding the quality assurance of cochineal dye products.

Full text of DOI:10.1248/cpb.c16-00838

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06
Chem. Pharm. Bull. 65, 306–310 (2017)
Vol. 65, No. 3
Note
Kinetic Study of the Equilibration between Carminic Acid and Its Two
Isomers Isolated from Cochineal Dye
a
a
a
b,c
c
Kazumasa Zaima, Azusa Fukamachi, Ryoko Yagi, Yusai Ito, Naoki Sugimoto,
c
a
,a
Hiroshi Akiyama, Kazufusa Shinomiya, and Naoki Harikai*
a
b
School of Pharmacy, Nihon University; 7–7–1 Narashinodai, Funabashi, Chiba 274–8555, Japan: Department of
s University; 2–2–1 Hitotsubashi, Chiyoda-
ku, Tokyo 101–8437, Japan: and National Institute of Health Sciences; 1–18–1 Kamiyoga, Setagaya-ku, Tokyo
58–8501, Japan.
Received October 25, 2016; accepted December 14, 2016
Food Science and Nutrition, Faculty of Home Economics, Kyoritsu Women
̕
c
1
Carminic acid (CA) is a major component of cochineal dye used in food additives, cosmetics, and phar-
maceuticals. CA and its isomers, 2-C-α-glucofuranoside and 2-C-β-glucofuranoside of kermesic acid (DCIV
and DCVII, respectively), were isolated from cochineal dye and the equilibrium constants (K) between CA,
DCIV and DCVII were investigated. DCIV was partially converted to CA and DCVII, and DCVII was con-
verted to CA and DCIV, whereas CA was very stable and only very slightly converted to DCIV and DCVII.
Most of the DCIV and DCVII was converted to CA under aqueous conditions. The kinetic rate constants (k)
ꢀ1
for the degradation of DCIV within the first day of incubation at 24°C was determined to be 0.901d and
ꢀ1
for the degradation of DCVII it was determined to be 1.102d . The k value for the formation of CA from
ꢀ1
the remaining DCIV was calculated to be 0.146d and for the formation of CA from the produced DCVII it
ꢀ1
ꢀ7
ꢀ3
ꢀ3
was found to be 0.148d . The K values were calculated as 1.22ꢁ10 , 2.61ꢁ10 and 2.36ꢁ10 mol/L for CA,
DCIV and DCVII, respectively. These findings will be helpful for ensuring the safety and for aiding the qual-
ity assurance of cochineal dye products.
Key words cochineal; kermesic acid; anomerization; glucofuranoside; kinetic rate constant; equilibrium con-
stant
Cochineal is a natural red dye extracted from Dactylopius of inter-conversion has not been clarified. The time course of
coccus Costa. Historically, this dye was used predominantly the transformation between CA, DCIV and DCVII has not yet
1)
for coloring artwork and textiles, but currently it is also been clarified. Knowledge of the time-dependent changes in
used worldwide in food additives, cosmetics and pharma- the minor components DCIV and DCVII is very important for
ceuticals. Cochineal dye is a generally highly safe material, establishing the safety and quality assurance of cochineal dye
but occasionally induces harmful allergic reactions including containing in foods and drugs.
2
–4)
anaphylaxis, urticarial and occupational asthma.
Causative
In this study, we purified CA, DCIV and DCVII from a
5
)
substances of the allergic reactions are not well-known, and cochineal dye product used as a food additive and determined
the involvement of minor components of the dye in the reac- the kinetic rate constant (k) and the equilibrium constants (K)
tions has still not been studied. In addition, the minor compo- for the interconversion of CA, DCIV and DCVII under equi-
nents in food additives, colored textiles and historical artwork librium conditions in aqueous solution.
generally have been required to identify the species and the
geographical origin of the cochineal by using multivariate Experimental
1
statistical methods such as principal components analysis,
General Experimental Procedures H-NMR spectra
partial-least squares discriminant analysis and hierarchical were measured using an ECA-800 spectrometer (JEOL Ltd.,
1,6–10)
cluster analysis.
Tokyo, Japan) and the chemical shifts were referenced to
The quality, insect species, and geographical origin of co- tetramethylsilane (TMS, δ =0ppm) as an internal standard.
H
chineal dye are typically evaluated using an HPLC equipped IR spectra were measured using an FT/IR-4100 spectrometer
6
,7,11,12)
with a UV-Vis detector
detector
and a photodiode array (JASCO, Tokyo, Japan) and UV spectra were obtained using
to determine carminic acid (CA), the main con- a V-630 spectrophotometer (JASCO). The high-resolution
stituent of cochineal, as well as minor components. electrospray ionization time-of-flight mass spectra (HR-ESI-
1
,13–15)
Minor components isolated from cochineal dye have TOF-MS) were obtained using Xevo G2 Q-Tof mass spectrom-
been identified as flavokermesic acid, kermesic acid, 2-C- eter (Waters, Milford, MA, U.S.A.) in negative mode, where
glucopyranoside of flavokermesic acid, 4-aminocarminic acid, the [M−H]− peak was indicated as m/z. Optical rotations
3
-O-glucopyranoside of flavokermesic acid, and 3,4-dideoxy- were measured with a P-1020 automatic digital polarimeter
11,14,15)
carminic acid.
The two isomers of CA were identified as (JASCO).
2
-C-α-glucofuranoside and 2-C-β-glucofuranoside of kermesic
Materials Cochineal dye was obtained from Alps Phar-
maceutical Industry (Gifu, Japan). The components were
14–16)
acid (DCIV and DCVII, respectively).
Stathopoulou et al. recently reported that DCIV and isolated and purified using reagent grade chemicals purchased
DCVII produce two isomeric forms via anomerization and from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
15)
both can be transformed to CA, although the mechanism
HPLC Conditions The analytical system comprised two
*
To whom correspondence should be addressed. e-mail: harikai.naoki@nihon-u.ac.jp
©
2017 The Pharmaceutical Society of Japan

Vol. 65, No. 3 (2017)
Chem. Pharm. Bull.
307
Fig. 1. Changes in the Concentrations of CA, DCIV and DCVII at 24°C (Left Side, (a)–(c)) and 40°C (Right Side, (d)–(f)), in Aqueous Solutions of
a) and (d) CA, (b) and (e) DCIV, and (c) and (f) DCVII
(
●: CA, ◆: DCIV, and ▲: DCVII.
LC-10ADVP pumps (Shimadzu, Kyoto, Japan), a SPD-10AVP 0.05% phosphoric acid–methanol (55:45) and detection: UV
UV-Vis detector, a CTO-10A column heater, and a Chromato- 276nm. The calibration curves for all three compounds (CA,
PRO integrator (Run Time Instruments, Kanagawa, Japan) DCIV and DCVII) were linear from 0.0016 to 1.0mg/mL
2
and the separation conditions were as follows: column: Inertsil (r >0.999).
ODS-4 (4.6×250mm, 5µm, GL Science, Tokyo, Japan), flow
The preparative system comprised two LC-10AS pumps
rate: 1.0mL/min, column temperature: 30°C, eluent: aqueous (Shimadzu), a SPD-10AV UV-Vis detector, a CTO-10A col-

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Chem. Pharm. Bull.
Vol. 65, No. 3 (2017)
Fig. 2. Kinetic Curves for the Degradation of DCIV and DCVII
Kinetic curves showing the degradation of DCIV and DCVII in DCIV aqueous solution (left side) and DCVII aqueous solution (right side) at 24°C, (a) DCIV in DCIV
aqueous solution, (b) DCVII in DCIV aqueous solution, (c) DCIV in DCVII aqueous solution and (d) DCVII in DCVII aqueous solution.
−1
umn heater, and a C-R8A integrator (Shimadzu) and the H-5). IR (KBr) cm : 3440, 2930, 1570. UV λ (H O) nm (ε):
max
2
separation conditions were as follows: column: Inertsil ODS-4 278 (37540), 313 (11950), 491 (8310). HR-ESI-TOF-MS m/z:
−
26
D
(
14×250mm, 5µm, GL Science), flow rate: 5.0mL/min, col- 491.0832 [Calcd for C H O (M−H) , 491.0826]. [α] −94.0
2
2
19 13
umn temperature: 40°C, eluent: aqueous 0.1% formic acid– (c=0.01, MeOH).
methanol (50:50) and detection: UV 254nm.
Isolation and Purification CA Cochineal dye consisted
DCVII
1
H-NMR (CD OD) δ: 2.78 (3H, s, CH ), 3.72 (1H, dd,
3
3
of 94.0% CA, 2.4% DCIV and 1.2% DCVII at their compo- J=11.5, 5.2Hz, H-6′b), 3.82 (1H, dd, J=11.5, 3.0Hz, H-6′a),
sitions, respectively, by the HPLC analysis. A part of large 4.01 (1H, ddd, J=8.4, 5.3, 3.0Hz, H-5′), 4.34 (1H, dd, J=3.4,
amounts of CA was isolated at the purity of 97.8% before elut- 1.3Hz, H-3′), 4.40 (1H, dd, J=8.5, 3.3Hz, H-4′), 4.52 (1H, dd,
ing DCIV and DCVII using the conventional column chroma- J=2.9, 1.0Hz, H-2′), 5.72 (1H, d, J=3.0Hz, H-1′), 7.73 (1H,
−1
tography described below.
s, H-5). IR (KBr) cm : 3450, 2920, 1630. UV λ
(H O) nm
max 2
DCIV and DCVII Cochineal dye (1.5g) dissolved in (ε): 278 (26800), 310 (9090), 489 (6160). HR-ESI-TOF-MS m/z:
−
26
D
water (1.5L) was subjected to reversed-phase column chroma- 491.0832 [Calcd for C H O (M−H) , 491.0826]. [α] −210.5
2
2
19 13
tography using a column (40×180mm, width×height) filled (c=0.02, MeOH).
with octadecylsilyl (ODS)-silica resin (YMC GEL ODS-A,
Kinetic Study Aqueous solutions of CA, DCIV and
2nm S-150µm; YMC, Kyoto, Japan). The DCIV- and DCVII were prepared at 2.0mmol/L. Each sample solution
1
DCVII-containing fractions were obtained using aqueous 10% was divided into aliquots and incubated at either 24 or 40°C
methanol after CA was eluted. After evaporating methanol for 0.5, 1, 2, 4, 8, 16 and 24d, then a portion of each sample
from each fraction under vacuum at room temperature, the was subjected to analytical HPLC.
residue was re-dissolved in an aliquot of water and then sub-
The k value was calculated according to the formula:
jected to preparative HPLC to obtain 11.1mg of DCIV (84.4% logC =−kt/2.303+logC0 (lnC =−kt+lnC ), where C cor-
t
t
0
t
purity) and 2.7mg of DCVII (88.7% purity). The purity of responds to the concentration of the sample at t days after
these compounds was calculated from their peak areas in the the start of the incubation, and C is the concentration of the
0
HPLC chromatogram, respectively. These purified compounds sample at the start of the incubation.
were then subjected to structural analysis and kinetic studies.
The obtained spectral data for DCIV and DCVII are described Results and Discussion
below:
Kinetic studies on CA, DCIV and DCVII were conducted
DCIV
after isolating the compounds from cochineal dye product.
H-NMR (CD OD) δ: 2.78 (3H, s, CH ), 3.67 (1H, dd, Figure 1 shows the changes in the concentrations of CA,
1
3
3
J=11.7, 5.6Hz, H-6′b), 3.79 (1H, dd, J=11.8, 3.5Hz, H-6′a), DCIV and DCVII in aqueous solution incubated at either 24°C
.01 (1H, dd, J=7.6, 2.7Hz, H-4′), 4.08 (1H, ddd, J=9.1, 5.6, (left side (a)–(c)) or 40°C (right side (d)–(f)). As shown in Fig.
.5Hz, H-5′), 4.23 (1H, dd, J=2.7, 1.0Hz, H-3′), 4.26 (1H, dd, 1(a), CA was converted very slowly to DCIV and DCVII over
J=3.0, 1.1Hz, H-2′), 5.36 (1H, d, J=2.9Hz, H-1′), 7.71 (1H, s, time at 24°C, with 0.012mmol/L DCIV and 0.019mmol/L
4
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Vol. 65, No. 3 (2017)
Chem. Pharm. Bull.
309
DCVII present in the aqueous CA solution after 24d. This re-
As described above, the degradation rates of DCIV and
−1
sult suggested that CA, DCIV and DCVII reached equilibrium DCVII in aqueous solution were similar (0.901d for DCIV
in the aqueous CA solution at 24°C. As shown in Fig. 1(b), and 1.102d− for DCVII). This result suggests that the anom-
DCIV was partially degraded during incubation at 24°C, with erization reaction from the α- to β-form and the β- to α-form
CA and DCVII produced within one day, and then DCIV and proceeds at similar rates after the dried purified sample is
DCVII were gradually converted to CA until equilibrium was dissolved in water when DCIV is 2-C-α-glucofuranoside form
1
established between the three compounds.
and DCVII is 2-C-β-glucofuranoside form, respectively. Fol-
Figure 2 shows kinetic curves for the degradation of DCIV lowing anomerization, both DCIV generated from DCVII and
and DCVII in each aqueous solution at 24°C. The k value DCVII generated from DCIV in aqueous solution are slowly
1
for the degradation of DCIV within the first day of incuba- converted into CA until equilibrium is achieved with the
tion at 24°C was determined to be 0.901d− (Fig. 2(a)), the remaining original DCVII or DCIV and this conversion into
1
−
1
k value for the formation of CA from the remaining DCIV CA occurs at a similar rate (in the DCIV solution: 0.146d
2
−1
−1
was calculated to be 0.146d (Fig. 2(a)), and the k value for for DCIV and 0.148d for DCVII, in the DCVII solution:
the formation CA from the produced DCVII was found to 0.109 d− for DCIV and 0.147d for DCVII). The dynamic
1
−1
−1
be 0.148d (Fig. 2(b)). Figure 1(c) shows that some DCVII equilibrium between CA, DCIV and DCVII in aqueous solu-
was degraded, and CA and DCIV were produced in the aque- tion is schematically illustrated in Fig. 3.
4
ous DCVII solution within one day. DCIV and DCVII were
The K value for CA in aqueous solution is about 10 fold
then gradually converted into CA until equilibrium was at- less than that for DCIV and DCVII, which suggests that CA
tained between these three compounds. The k value for the is more stable than DCIV and DCVII. CA contains a gluco-
1
degradation of DCVII within the first day of incubation was pyranose moiety, whereas DCIV and DCVII contain a gluco-
determined to be 1.102d− (Fig. 2(d)), the k value for the furanose moiety. It has been reported that glucose is more
formation of CA from the produced DCIV was calculated as stable in the pyranose form than in the furanose form because
1
−
1
17)
0
.109 d (Fig. 2(c)), and the k value for the formation of CA of the lower energy of its intermolecular hydrogen bonds,
2
−1
from the remaining DCVII was 0.147d (Fig. 2(d)). From the and that in aqueous solution, glucose primarily exists in the
results shown in Figs. 1(a)–(c), the K values were calculated to pyranose form and not in the furanose form (≤1%) at equilib-
−
7
−3
18)
be 1.22×10 mol/L for CA, 2.61×10 mol/L for DCIV, and rium. These observations may explain the difference in the
−3
2
.36×10 mol/L for DCVII, as summarized in Table 1.
At 40°C, DCIV (Fig. 1(e)) and DCVII (Fig. 1(f)) in aqueous DCVII.
solution both transformed into CA more rapidly than at 24°C This study focused on assessing the equilibrium between
and reached equilibrium within 2d, where it was difficult to C-glucofuranosides, reported only once previously, although
illustrate the figure on the kinetic curves. These results sug- there are a couple of reports on C-glycosides, including C-
equilibration rate between CA and its two isomers, DCIV and
19)
2
0)
21)
22)
gested that the rate of the equilibrium reaction depends on the glycoside flavonoids, xanthones and anthranoids. The k
temperature.
and K values calculated in the present study may help clarify
the structural changes that take place during anomerization
between C-glucopyranoside and C-glucofuranoside.
Table 1. Equilibrium Constants for Each Reaction at 24°C
Conclusion
Reaction
K (mol/L)
CA and its two isomers, DCIV and DCVII, were isolated
in highly purified form from cochineal dye using a series of
reversed-phase chromatographic methods and their kinetic
properties were evaluated. The degradation rates of DCIV
7
3
3
CA→DCIV+DCVII
DCIV→CA+DCVII
DCVII→CA+DCIV
1.22×10−
−
2.61×10
2.36×10−
Fig. 3. Schematic Illustration of the Dynamic Equilibrium between CA, DCIV and DCVII in Aqueous Solution

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Chem. Pharm. Bull.
Vol. 65, No. 3 (2017)
Akiyama H., Ohta K., J. Allergy Clin. Immunol. Pract., 1, 197–199
2013).
González M., Méndez J., Carnero A., Lobo M. G., Afonso A., J.
Agric. Food Chem., 50, 6968–6974 (2002).
Méndez J., González M., Lobo M. G., Carnero A., J. Agric. Food
Chem., 52, 1331–1337 (2004).
and DCVII were similar in aqueous solution. The DCIV and
DCVII produced in aqueous solution were slowly converted
into CA until equilibrium was reached, and the remaining
DCVII and DCIV were converted to CA at similar rates. The
(
6
7
)
)
4
K for CA was about 10 fold less than that for DCIV and
DCVII. These results suggested that CA is major component
in every kind of raw material, and the influence to the quality
and safety of products including cochineal dye is negligible
even if DCIV and DCVII are contained much. This study
8
)
Chávez-Moreno C. K., Tecante A., Fragoso-Serrano M., Pereda-
Miranda R., Biochem. Syst. Ecol., 38, 671–679 (2010).
9) Serrano A., Sousa M., Hallett J., Simmonds M. S., Nesbitt M.,
Lopes J. A., Analyst, 138, 6081–6090 (2013).
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van den Berg K. J., Anal. Chim. Acta, 897, 116–127 (2015).
components DCIV and DCVII will be very helpful for es-
tablishing the safety of cochineal dye products and for their
quality assurance.
1
1
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Acknowledgment This study was supported in part by a
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