Hydrophilic carotenoids: Surface properties and aggregation behavior of the potassium salt of the highly unsaturated diacid norbixin

Article abstract of DOI:10.1002/hlca.200900042

The oft-claimed 'good' water solubility of the food color norbixin (3) could not be confirmed. In contrast, the potassium salt 5 of norbixin formed suitable dispersions. The surface and aggregation properties of salt 5 were investigated and compared with

Full text of DOI:10.1002/hlca.200900042

Helvetica Chimica Acta – Vol. 92 (2009)
1741
Hydrophilic Carotenoids: Surface Properties and Aggregation Behavior of the
Potassium Salt of the Highly Unsaturated Diacid Norbixin
by Stefanie Breukers, Christer L. Øpstad, Hans-Richard Sliwka, and Vassilia Partali*
Department of Chemistry, Norwegian University of Science and Technology, NO-7491 Trondheim
(e-mail: vassilia.partali@chem.ntnu.no)
The oft-claimed ꢀgoodꢁ water solubility of the food color norbixin (3) could not be confirmed. In
contrast, the potassium salt 5 of norbixin formed suitable dispersions. The surface and aggregation
properties of salt 5 were investigated and compared with other naturally occurring and synthetic
hydrophilic carotenoids (Table).
Introduction. – Considered in a historical perspective, two carotenoids out of the
known ca. 750 have been abundantly used since ancient times: crocin (1) and bixin (2)
[1 – 3]. These two carotenoids are highly unsaturated diacid derivatives: crocin (1) is a
naturally occurring sugar ester, and bixin (2) (Scheme) is a monomethyl ester (crocin ¼
bis(6-O-b-d-glucopyranosyl-b-d-glucopyranosyl)-8,8’-diapo-y,y-carotenedioate ¼ 6-
O-b-d-glucopyranosyl-b-d-glucopyranose 1,1’-[(2E,4E,6E,8E,10E,12E,14E)-2,6,11,15-
tetramethylhexadeca-2,4,6,8,10,12,14-heptaenedioate]; bixin ¼ 6-methyl 6’-hydrogen
9-cis-6,6’-diapo-y,y-carotenedioate ¼ 1-methyl 20-hydrogen (2E,4Z,6E,8E,10E,12E,
14E,16E,18E)-4,8,13,17-tetramethyleicosa-2,4,6,8,10,12,14,16,18-nonaenedioate). Both
compounds are frequently used as food colors. Crocin (1) is highly soluble in H2O;
there is practically no saturation point [4]. Bixin (2) is H2O insoluble. Hydrolyzing
bixin (2) gives norbixin (¼6,6’-diapo-y,y-carotenedioic acid; 3), which is often
presented in the literature as H2O-soluble, e.g., it is claimed that solutions up to 5% can
be achieved [5 – 8]. Crocin (1) and norbixin (3) are therefore often identified as the two
outstanding H2O-soluble compounds in the range of the otherwise hydrophobic
carotenoids. However, there is no plausible reason for claiming that the longer-chain
diacid norbixin (3; C24) is H2O soluble, when the shorter-chain crocetin (4; C20) has
been found to be nearly insoluble in H2O [9]. Consequently, other authors are more
definitive and affirm that the Na or K salts of norbixin are the true H2O-soluble
compounds [2][10].
The dipotassium salt 5 of norbixin is composed of a hydrophobic polyene chain
connected to hydrophilic groups at both ends. This structural characteristic confers to
the molecule the character of a bolaamphiphile, showing surfactant activity and the
tendency to form aggregates in H2O. Besides its use as food color (E160b), norbixin (3)
and its Na or K salts (see 5) react as antioxidants and singlet-oxygen quenchers, and
demonstrate other biological activities [7][11 – 13].
In spite of the widespread commercial applications of norbixin and its salts, the
surfactant data and aggregation properties of these biodegradable bolaamphiphiles has
ꢂ 2009 Verlag Helvetica Chimica Acta AG, Zꢃrich

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Helvetica Chimica Acta – Vol. 92 (2009)
Scheme

Helvetica Chimica Acta – Vol. 92 (2009)
1743
not been investigated. The now presented results are an extension of our investigations
of natural and synthetic hydrophilic carotenoids [4][14 – 16].
Results and Discussion. – Synthesis of Norbixin (3). Bixin (2) was hydrolyzed in
BuOH with 25% KOH/MeOH at 1108 for 7 h. Although high temperature favors
isomerization to the more stable all-trans-configurated norbixin (3), a mixture of 3 and
its cis-isomer was obtained (VIS spectrum: cis-peak at 350 nm) [17]. Since it appeared
difficult to separate these isomers by column chromatography, the mixture was
isomerized by adding some drops of a methanolic I2 solution [18]. After workup and
freeze-drying, pure trans-configurated 3 was collected (Scheme).
Aggregation Behavior of Norbixin (3). A few drops of MeOH were given to
norbixin (3) prior to adding H2O to facilitate dispersion formation. After stirring one
week under N2 at 208, the dispersibility of 3 was determined spectroscopically to 0.2%.
It was found that 3 appeared predominantly as H-aggregates [19] in H2O, which
strongly suggests that even at this low concentration, an aggregate dispersion is present
and not, as has been stated, a solution. The critical H2O concentration in MeOH for
aggregation is 62% (Fig. 1). Large aggregates were formed with a hydrodynamic radius
rH ꢀ 2.3 mm. The low hydrophilicity of norbixin (3) prevented tensiometric measure-
ments. The hydrophilicity, however, was high enough to inhibit the preparation of
surface monolayers [20].
Fig. 1. UV/VIS Spectra of norbixin (3) as a function of solvent concentration. In H2O lmax 384 nm, in
MeOH lmax 450 nm. With gradual addition of H2O to the MeOH solution, aggregates are formed; in
contrast, with gradual addition of MeOH to the aqueous dispersion, the aggregates are disrupted. The
critical solvent concentration for aggregation is 62% H2O in MeOH; for disruption, 38% MeOH in H2O.

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Helvetica Chimica Acta – Vol. 92 (2009)
Surface Tension and Critical Aggregate Concentration of the Potassium Salt 5 of
Norbixin (3). Since the hydrophilicity of acid 3 is too low for practical purposes,
potassium salt 5 of norbixin was prepared. Salt 5 was dispersible in H2O to 8% and
formed H-aggregates (Fig. 2) with rH ꢀ 50 nm. Rotational peaks in the intensity plot of
the particle-size analyzer indicated the occurrence of nonspherical aggregates. The
surface tension g for various concentrations (c) of 5 in H2O was determined with a
tensiometer. A plot of g vs. ln c gave, at the point of discontinuity, the critical aggregate
concentration cM ¼ 0.0004 mol/l (¼0.16 mg/ml ꢀ 0.02%); at this point, the surface
tension was gc ¼ 56 mN/m (Table). The surface-tension measurements were preceded
by long equiliMbrium periods (>20 min) each time the g of a new concentration was
measured (Fig. 3). The values were recorded after 25 min and are, therefore,
considered to be indicative rather than definite.
Fig. 2. UV/VIS Spectra of potassium salt 5 of norbixin in H2O (lmax 415 nm) and MeOH (lmax 445 nm)
Table. Selected Data for Potassium Salt 5 of Norbixin (3) and Related Compounds
Norbixin (3)
Potassium salt 5
Crocin (1) [4]
CardaxTMa
)
Astalysineb)
Sol./disp. [%]
0.2
8
56
no saturation
52
0.9
60
18
58
gc [mN/m]
M
pc [mN/m]
16
21
13
14
M
cM [mm]
0.36
0.73
230
0.82
1.4
115
0.45
0.7
240
2.18
0.7
240
G 10ꢁ6 [mol j m2]
am [ꢄ2]
DGag [kJ/mol]
DGad [kJ/mol]
DGadꢁag [kJ/mol]
KM
Kad
Kadꢁag
ꢁ 59
ꢁ 82
ꢁ 23
2900
61000
22
ꢁ 17.5
ꢁ 32.5
ꢁ 15
1200
450000
370
ꢁ 54.8
ꢁ 73.6
ꢁ 18.8
1800
23000
13
ꢁ 90.4
ꢁ 109.7
ꢁ 19.3
1500
7200
5
AMER
1.4
1.8
1.3
1.2
a) Data taken from [15]. b) Data taken from [16].

Helvetica Chimica Acta – Vol. 92 (2009)
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~
Fig. 3. Change of surface tension g for potassium salt 5 of norbixin in H2O with time. , c ¼ 98 mg/l (left
^
axis); , c ¼ 1000 mg/l (right axis).
The surface excess concentration G was calculated with the equation G ¼ (ꢁ1/
nRT) · (dg/d(ln c)) ¼ (ꢁc/nRT) · (dg/dc) with n ¼ 3, assuming full dissociation of the
salt in H2O. The measured low surface concentration G ¼ 0.7 · 10ꢁ6 mol/m2 corresponds
to a high molecule area am ¼ 230 ꢄ2, which can be associated with horizontally lying
molecules of 5 at the water surface with both end groups anchored in the H2O. Other
ionic bolaamphiphilic carotenoids behave similarly (Table). With the values of cM, g, G,
am, and the surface pressure pcm, the free energy of adsorption DG8 and aggregation
ad
DG8 as well as the equilibrium constants for aggregation and surface adsorption were
ag
calculated. DG8 was derived from the equation DG8 ¼ nRTln cM þ 2RTbln 2 [21].
ag
ag
Again, the prefactor n denotes the number of dissociated species in H2O – similar to
other carotenoid salts with low cM (Table), we assumed full dissociation of 5 and set
n ¼ 3 [15][16]. The free energy of adsorption was calculated with DG8 ¼ DG8 ꢁ 6.023 ·
ad
ag
10ꢁ3. The equilibrium constants reflect the high preference of the molecules of 5 to be
adsorbed at the water surface. The adsorption-micellar energy ratio (AMER) [22]
DG8 /DG8 has been proposed as a surfactant-performance indicator. AMER Values
ad
ag
close to unity imply dense monolayer formation, enhanced micelle concentration, and
high ability in flotation, cleaning, and wetting. The AMER value for salt 5 is similar to
other ionic carotenoid surfactants.
Conclusion. – The potassium salt 5 of norbixin in H2O formed clear yellow
dispersions and showed the typical surface and aggregation properties of (ionic)
carotenoid bolaamphiphiles. Norbixin (3) was found to be almost insoluble (as well as
indispersible) in H2O, in contrast to previous statements in the literature. Crocin (1)
remains as the sole naturally – and abundantly occurring – carotenoid exhibiting true
H2O solubility.

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Helvetica Chimica Acta – Vol. 92 (2009)
Experimental Part
1. Norbixin (3). Bixin (2) was hydrolyzed by modifying a previous procedure [23]. Thus, 2 (0.1 g,
0.2535 mmol) was dissolved in 25% KOH/MeOH (0.25 ml), and BuOH (5 ml) was added. The soln. was
heated to 1108 under N2 for 7 h (TLC monitoring (silica gel sheets, acetone/hexane 1.5 :1)). After solvent
evaporation, H2SO4 (1.25 ml, 25%) was added and the soln. stirred for 5 min. Then, H2O (5 ml) was
added, and the mixture stirred for 30 min at r.t. CH2Cl2 (5 ml) was added, and the org. phase washed
several times with H2O until neutrality and then concentrated. The product was dissolved in acetone and
dried (Na2SO4). TLC and subsequent VIS-spectra analysis showed the occurrence of a cis-isomer (cis-
peak at 350 nm). The isomers were dissolved in MeOH and several drops of a I2/MeOH soln. were added,
which catalyzed cis to trans isomerization. After freeze-drying, 3 (29 mg, 30%) was obtained. VIS: Fig. 1.
1H-NMR (400 MHz, CD3OD): 7.85 (d, HC¼CHCOOH); 7.3 – 6.2 (m, CH of the polyene); 6.1 – 5.8 (m,
CH¼CHꢁCOOH); 2.1 – 1.9 (4 Me) [7]; no signals for the cis-isomer and for MeO of bixin (2). ESI-MS:
403 ([380 þ Na]þ) [17].
2. Norbixin Dipotassium Salt (¼(2E,4E,6E,8E,10E,12E,14E,16E,18E)-4,8,13,17-Tetramethyleicosa-
2,4,6,8,10,12,14,16,18-nonaenedioic Acid Potassium Salt (1:2); 5). Norbixin (3; 40.5 mg, 0.1064 mmol)
was dissolved in CH2Cl2/MeOH 1:0.6 (60 ml) at 08, and the same mol amount of 25% MeOK/MeOH
(63 ml, 0.2134 mmol) for each COOH group was added. The solvents were evaporated without heating.
VIS: Fig. 2.
3. Aggregation. Aggregate behavior was monitored by dispersing a known amount of 3 with H2O in a
UV cell. Several ml of MeOH were added prior to adding H2O to assure full dispersion. MeOH quantities
(150 ml) were continuously added, and the spectra recorded until the aggregates were disrupted forming a
monomolecular soln. Similarly, to a specified amount of 3 dissolved in MeOH, H2O (150 ml) was
gradually added until the aggregation peak was observed. Both measurements gave similar results
(Fig. 1).
4. Dispersibility. Dispersibility of 3 and 5 was determined spectroscopically in H2O (Milli-Q).
5. Particle Size. Aggregate size was determined with a N5 submicron-particle-size analyzer (by PCS;
Beckman Coulter, Inc., Fullerton) at angles presenting reliable values after filtering the dispersion with a
200-nm filter.
6. Surface Parameters. Critical aggregate concentration and surface tension were determined in a
conical, Teflon-coated vessel with a Wilhelmy plate on a Krꢀss-K100 tensiometer by gradually adding
H2O to an aq. dispersion of 5 with a Metrohm-765 dosimat. The g value was taken 25 min after each
dilution (Fig. 3). The measurements were made in duplicate at different times by different operators. For
the calculation of thermodynamic data, see [4].
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Received February 6, 2009