Properties of red pigments prepared fromgeniposidic acid and amino acidsNobuharu Moritome,1* Yuki Kishi1 and Satoshi Fujii21Research Laboratory, Taito Co Ltd, 1-26 Higashi Shiriike-Shinmachi Nagata-ku, Kobe 653-0023, Japan2Faculty of Agriculture, Kobe University, Nada-ku, Kobe 657-0013, Japan
Abstract: Red pigments formed from geniposide and amino acids were studied. Geniposide, the
principal iridoid glucoside contained in the fruit of Gardenia jasminoides Ellis, was hydrolysed to
geniposidic acid as a precursor for the pigment. The pigments prepared anaerobically with arginine or
glutamic acid under acidic conditions with a large excess of citric acid, were reddish purple and had
properties governed by the electrostatic character of the amino acid. Relative molecular masses of
these pigments were determined to be several thousand by gel ®ltration. The pigments showed good
stability to heat and pH, but were gradually bleached by light. The safety of the pigments was evaluated
by studing their oral acute and the 5-month oral toxicity of arginine-red pigment using mice. The
pigments were considered to be of low toxicity.
# 1999 Society of Chemical Industry
Keywords: red pigment; geniposidic acid; geniposide; amino acid; gardenia iridoids; food colourants
INTRODUCTIONGardenia (Gardenia jasminoides Ellis) is an evergreen
shrub cultivated in temperate regions of China,
Taiwan and the Philippines and bears reddish-yellow
oval ripening fruits in the late autumn. Gardenia fruits
contain yellow carotenoids, crocin and its congeners,
and nine iridoid glucosides including geniposide (GS,
Fig 1) as the main constituent and geniposidic acid
(GSA, Fig 1).1±7 Genipin, the aglycone of GS, reacted
with methylamine to give several intermediary brown-
ish-red pigments, which polymerised under aerobic
conditions to yield a blue pigment.8,9 Recently, the
pigment has been used as a valuable blue food
colourant in Japan. Thus iridoid glucosides, obtained
industrially from gardenia fruits, have been converted
into the blue food colourants under aerobic conditions
by food additive grade enzymes or micro-organisms
containing b-glucosidase in the presence of hydrolysed
proteins and amino acids.10 The properties and
toxicological safety of the blue pigment were reported
by Touyama et al.11 A pigment, prepared from GSA,
instead of GS as the raw material, was purple.12 We
obtained an even more reddish pigment by optimisa-
tion of the conditions of formation. This paper reports
on the properties of the red pigment produced from
GSA with arginine or glutamic acid.
METHODS AND MATERIALSMaterialsExtract of gardenia fruits was obtained. An ethanol
extract of crushed gardenia fruits (2.5kg) was evapo-
rated to remove the alcohol under reduced pressure
and diluted with water to 2l. The aqueous solution was
extracted with ethyl acetate to remove the less polar
substances and the aqueous layer was concentrated
under reduced pressure. This solution was applied to
an Amberlite XAD-7 column (2.5 l; Organo, Tokyo,
Japan) and eluted with water, 20% ethanol, 50%
ethanol and ethanol successively. Fractions containing
GS were collected and evaporated in vacuo. The
resulting solution was applied to a chromatography
column, using Diaion HP-20 (1.5 l; Mitsubishi
Chemical, Tokyo, Japan) as the absorbent, and eluted
with water and 50% ethanol. The puri®ed extract
(187g; GS = 51.3%, determined by HPLC) was
obtained by evaporating appropriate fractions to
dryness under reduced pressure.
Cellulase Y-NC, originating from Aspergillus niger,was purchased from Yakult Pharmaceutical Ind
Figure 1. Geniposide and geniposidic acid.
Journal of the Science of Food and Agriculture J Sci Food Agric 79:810±814 (1999)
* Correspondence to: Nobuharu Moritome, Research Laboratory, Taito Co Ltd, 1-26 Higashi Shiriike-Shinmachi Nagata-ku, Kobe 653-0023,Japan.(Received 4 July 1997; revised version 10 August 1998; accepted 7 October 1998)
# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50 810
(Tokyo, Japan). Amino acids were purchased from
Ajinomoto (Tokyo, Japan). Glucosylated rutin13 was
purchased from Toyo Sugar Re®ning (Tokyo, Japan).
Other reagents were obtained from Nacalai Tesque
(Kyoto, Japan). Citric acid was used as the mono-
hydrate. Deionised water was used throughout.
Preparation of the red pigment from arginineA 35g portion of the puri®ed extract was hydrolysed to
crude GSA with 100ml of 2.5M NaOH at 60°C for 1h
and adjusted to pH 4 with citric acid. After removal of
cations by passage through a column of 100ml
Amberlite IR-120B (H�), 9.4g of arginine was added
to the eluent. The resulting solution was adjusted to
pH 4 with 10M NaOH and diluted to 300ml with
water. Enzymic hydrolysis of GSA was carried out with
2.5g of cellulase powder at 50°C for 20h in an argon
atmosphere. The reaction mixture was heated at 90°Cfor 2h and centrifuged at 18,500� g for 10min. For
removal of the excess of ionic components, such as
arginine and citric acid, the supernatant solution was
passed through a column of Amberlite IR-120B (H�)
(100ml) and IRA-63 (OHÿ) (200ml). An appropriate
amount of dextrin was added to the eluent as a bulking
agent to adjust the colour intensity to A = 500 at
535nm on a dry-weight basis. The solution was spray-
dried and pulverised with a Pulvis Mini-Spray, model
GB-21 (Yamato Scienti®c Co Ltd, Tokyo, Japan) to
yield the pigment powder (PRA, 75g), which was used
in subsequent experiments.
Preparation of the red pigment from glutamic acidThe crude GSA solution was prepared as above but
without the ion-exchange resin treatment. Glutamic
acid (8.4g) and citric acid (11.3g) were added to
about 100ml of the crude GSA solution and adjusted
to pH 4.5 with 10M NaOH and diluted to 300ml with
water. Enzymic hydrolysis of GSA, heating and
centrifuging were carried out as above. Next, the
supernatant solution was diluted to 600ml with water
and concentrated to 200ml with an ultra®ltration
system (Tosoh, Tokyo, Japan). The system consisted
of a membrane module (UF-LMS), pump (LP-3000)
and membrane cassette (UF-10PS MW cut-off
10,000, 200cm2� 3 sheets). The ultra®ltration was
repeated twice. The puri®ed solution was converted
into a powder and the colour intensity adjusted to
A = 500 at 532nm as in the preparation of PRA. The
powdered pigment (PRG, 119g) was used in subse-
quent experiments.
Measurement of absorbance and colourThe pigment obtained was characterised by its
absorbance at lmax and its hue after appropriate
dilution with water or buffer. Absorbance of the
diluted solution was measured in the visible region
on a UV-265FW spectrophotometer (Shimadzu,
Kyoto, Japan), using a 1cm cuvette against a water
blank. After adjusting the absorbance at lmax to 0.5,
the colour of the solution in Hunter's `L-a-b' system
was measured by an SZ-S80 II colorimeter (Nippon
Denshyoku Kougyou, Tokyo, Japan) with transmitted
light.
Characterisation of PRA and PRGSolubility
A 0.5g portion of PRA or PRG was added to 5ml of
water, propylene glycol, ethyl acetate, hexane, ethanol
and 250, 500 and 750gkgÿ1 mixtures of aqueous
ethanol and aqueous propylene glycol. Each suspen-
sion was sonicated with a Bransonic cleaner (Model
B-2200, Connecticut, USA) for 10min at room
temperature. After centrifugation of the mixtures at
1600� g for 10min, 1ml of each supernatant was
diluted with water to 50ml and the absorbance was
measured. The solubility of the pigment in each
solvent was calculated by dividing the absorbance of
the resultant supernatant by the original absorbance.
Effect of pH on the character of the pigment
To avoid the effect of citric acid, phosphate buffer was
used. Phosphate buffers in the range of pH 2±4 were
adjusted with phosphoric acid. Basic solutions of pH
7±12 were adjusted with aqueous 5M NaOH. PRA or
PRG, respectively, was dissolved in each pH solution
and the absorbance was adjusted to around two. After
standing for 1h at room temperature, each solution
was centrifuged at 1600� g for 10min. The absor-
bance of the supernatant was measured.
Light stability
PRA or PRG, respectively, was dissolved in pH 3±7
McIlvaine's buffer to give A = 2 and placed into a
transparent glass bottle of 50ml volume. These bottles
were exposed to xenon light in a xenon-fade testing
instrument FAL-25AX (Suga Test Instruments,
Tokyo, Japan) at 40°C for 15h. Light stability was
determined by comparing the pre- and post-test
absorbance.
Thermostability
Sample solutions of PRA or PRG were prepared in a
similar manner as for the light stability test and heated
at 90°C for 30min. Thermostability was determined
by comparing the pre- and post-test absorbance.
Effect of antioxidants
Sodium ascorbate or glucosylated rutin was added in
the range of 50±2500mg litreÿ1 to pH 4 McIlvaine's
buffer containing PRA or PRG to give A = 1. The
light- and thermo-stabilities were examined as de-
scribed above and the effect of the antioxidants
evaluated by comparing the absorbance of the solu-
tions with and without added antioxidant.
Coprecipitation
PRA or PRG was dissolved in water to give A = 2. The
solutions were mixed in various proportions and
allowed to stand for 30min at room temperature.
After centrifugations at 7740� g for 5min, the
J Sci Food Agric 79:810±814 (1999) 811
Red pigments prepared from geniposidic acid and amino acids
absorbances of the supernatants were measured. In
addition, PRA or PRG was dissolved in aqueous 6M
guanidine hydrochloride to give A = 2. Equal volumes
of these solutions were mixed and the mixture was
allowed to stand for 2h. After centrifugation at
7740� g for 10min, the absorbance of the super-
natant was measured.
Relative molecular mass
Relative molecular masses of PRA and PRG were
estimated by gel ®ltration chromatography. Retention
times of the peaks of the pigments and markers were
determined. The relative molecular masses of the
samples were determined from a curve of retention
time versus logarithm of molecular mass for a standard
set of markers. The sample solutions were injected into
the Tosoh HPLC system (pump: CCPE, column:
TSK-GEL G3000SWXL with guard column, 7.8mm
id� 300mm, detector: RI-8000). The elution was
carried out with a solution containing guanidine
hydrochloride (6mol) and Na2HPO4 (50mmol) per
litre (pH 7.0) at a ¯ow rate of 0.5ml minÿ1. The
eluents were measured at 535nm for the red pigments
and at 220nm or 280nm for the molecular mass
standards, ie, egg albumin (molecular mass 45,000),
horse heart-cytochrome C (12,400), human-ANP
(3080), human-angiotensin I (1296) and phenyl-
alanine (165).
Alternatively, PRA or PRG was dissolved in water or
HPLC solvent to give A = 5. A 0.4ml portion of each
of these solutions was placed in a centrifugal ®lter
device (Ultrafree-0.5 Biomax-10: nominal molecular
weight limit (NMWL) = 10,000; ÿ50: NMWL =
50,000; ÿ100: NMWL = 100,000, Nihon Millipore,
Tokyo, Japan) and centrifuged at 7740� g for 15min.
Subsequently, each ®lter device was washed by
spinning with 0.4ml of the same solvent at 7740� gfor 30min. The ®ltrate and the washing were
combined and diluted to 1ml with the same solvent.
The proportion of the pigment ®ltered through each
®lter was determined from the ratio of the absor-
bances. These experiments were repeated three times
and the results were averaged.
Safety Evaluation
Acute oral toxicity evaluations of arginine-red pigment
and glutamic acid-red pigment were performed in 5
week-old ddy mice. Mice were allocated to four
groups, each consisting of 10 males and 10 females.
The dose levels of aqueous arginine-red pigment
concentrated to A = 771 (speci®c gravity = 1.153) at
535nm were 0, 0.32, 0.40 and 0.50ml per 10g body
weight. Aqueous glutamic acid-pigment, which had
been concentrated to A = 2267 (speci®c gravity =
1.220) at 532nm, was administered in the same
volumes as arginine-red pigment. The single admin-
istration of aqueous pigment was carried out by gastric
tube and the mice were killed and examined after 2
weeks.
A 5-month oral toxicity evaluation of the arginine-
red pigment was performed in 5-week-old male and
female B6C3F1 mice. The dose levels of aqueous
pigment concentrated to A = 1061 at 535nm were 0,
0.5, 1.5 and 4.5% in the basal diets. The diets were
freely available throughout the test period. Mice were
allocated to four groups, each consisting of 12 males
and 12 females. After 21 weeks, all mice were killed
and their principal organs (heart, lung, spleen, liver,
kidney, adrenal, sexual gland and stomach) were
weighed. The histopathology was examined by micro-
scope.
Mutagenicity was evaluated by the Ames test with
the aqueous pigment solutions used for the acute
toxicity evaluation. The tests were carried out using
®ve strains of bacteria (Salmonella typhimurium TA98,
TA100, TA1535, TA1537 and Escherichia coli WP2
uvrA), with and without S9 microsomal activation
mixture and at ®ve dose groups with 5mg per plate as
the highest dose.
RESULTS AND DISCUSSIONRed pigment produced from arginine or glutamicacidAccording to preliminary experiments, arginine as a
basic amino acid and glutamic acid as an acidic amino
acid were chosen as starting materials for the produc-
tion of red pigment. The glutamic acid-pigment
formed no precipitate during the reaction, but the
arginine-pigment formed a large quantity of precipi-
tate during the heating process. PRA and PRG
powdered with dextrin showed similar visible spectra
with lmax 535nm and 532nm in water, respectively.
Hunter's L-a-b values were 71.6, 34.6, ÿ8.2 for PRA
and 72.6, 36.8, ÿ8.3 for PRG.
Characterisation of PRA and PRGSolubility
Both PRA and PRG were soluble in water, but in-
soluble in organic solvents, such as methanol, ethanol,
ethyl acetate and hexane. With increasing water
content, these pigments gradually dissolved in aque-
ous methanol, ethanol and propylene glycol. For
example, they were freely soluble in 250gkgÿ1
aqueous ethanol, but only sparingly soluble in
750gkgÿ1 aqueous ethanol. On the other hand, they
were readily soluble in 750gkgÿ1 aqueous propylene
glycol, but only sparingly soluble in neat propylene
glycol.
Effect of pH on the character of the pigment
Unlike anthocyanins and anthraquinones, PRA and
PRG did not show any signi®cant change in colour
tone and absorbance in the range of pH 3±8, except for
the slight turbidity produced by PRA at pH 8 and PRG
at pH 3.
Solutions of PRA and PRG in phosphate buffer
produced a precipitate and turbidity in basic or acidic
conditions, respectively (Fig 2). These phenomena
should be related to the pI value of the constituent
812 J Sci Food Agric 79:810±814 (1999)
N Moritome, Y Kishi, S Fujii
amino acid, ie arginine (pI 11.15) and glutamic acid
(pI 3.22). The effect of pI, however, was not sharp.
Light stability
PRA solutions in McIlvaine's buffer were turbid at
every pH, particularly above pH 5. Therefore it was
not possible to evaluate the light stability of PRA
exactly, but the maximum stability was observed at pH
4, with 88% of the absorbance remaining after the 15h
light exposure. On the contrary, PRG solutions were
more stable as pH increased. Thus, 75% of the
absorbance remained at pH 8, but only 21% at pH 3
due to precipitation. On light exposure, the visible
spectra of PRA and PRG solutions showed hypso-
chromic shifts at all pH values and their blue colour
tones were weakened.
Thermostability
Most PRA solutions could not be evaluated due to the
turbidity, except at pH 3 and 4, where hardly any
turbidity formed and the relative absorbance increased
by ca 16%. The relative absorbance of PRG solutions
increased by 16% at every pH, except that of the test
solution at pH 3, which decreased in absorbance due
to pigment precipitation. The blue tone was slightly
diminished in both pigment solutions at all pHs.
Effect of antioxidants
To reduce the deterioration of natural colourants,
antioxidants such as commercial ascorbic acid and
glucosylated rutin are frequently used in Japan. In the
light exposure, addition of glucosylated rutin to PRA
and PRG solutions inhibited the hypsochromic
change, that is, the increase in yellow tone, decrease
in red tone, depending on the dose added.
Addition of sodium ascorbate, however, above
250mg litreÿ1 resulted in an enhancement of the
absorbance without a diminution of the blue tone. In
the heating test, deepening of test solutions was
prevented by sodium ascorbate, but not by glucosy-
lated rutin. Consequently, the use of antioxidants
needs to be appropriately controlled.
Coprecipitation
A water-insoluble precipitate was formed by the
mixing PRA and PRG. Fig 3 shows the absorbance
of the supernatant of mixtures of the two pigments in
different proportions. The maximum amount of
precipitate was formed and the minimum absorbance
of the supernatant was observed, when equal amounts
of the two pigments were mixed, as judged by their
absorbance at lmax. This result suggested that the
numbers of positive charges per unit absorbance on
one arginine-pigment moiety equalled the negative
charges on one glutamic acid-pigment moiety. On the
contrary, no turbidity or precipitate was observed in
6M guanidine hydrochloride solutions of the pig-
ments. PRA, being a basic pigment due to its
guanidino group, was precipitated when mixed with
polyanionic compounds, such as PRG, citric acid and
polyphenols, eg tannins.
Relative molecular mass
From the results of gel ®ltration, the mean relative
molecular mass of PRA and PRG was estimated to be
roughly 3000 and 4500, respectively. The ultra®ltra-
tion method, however, showed the nominal molecular
mass to be extremely large (Table 1). The difference
between the methods was assumed to be caused by
self-association of the pigment. In presence of guani-
dine hydrochloride, a common dissociation agent for
proteins, ultra®ltration of the pigments proceeded
more easily (Table 1). Further, the relative molecular
mass of PRA was smaller than that of PRG in
agreement with the gel ®ltration results.
Safety Evaluation
In an acute toxicity evaluation, all mice treated with
arginine- or glutamic acid-pigment survived until the
Figure 2. Effect of pH on the solubility of PRA and PRG. PRA or PRG wasdissolved in aqueous solutions of pH 2–12 to give A = 2 at lmax. Afterstanding for 1h, each solution was centrifuged at 1600� g for 10min andthe absorbence of the supernatant was measured.
Figure 3. Coprecipitation from the mixed solution of PRA and PRG. PRAand PRG were dissolved separately in water or aqueous 6M guanidinehydrochloride to give A = 2 at lmax. The test solution was prepared bymixing the PRA and PRG solutions in various proportions and allowingthem to stand for 30min at room temperature. After centrifugation at7740� g for 5min, the absorbance of the supernatant was measured.
J Sci Food Agric 79:810±814 (1999) 813
Red pigments prepared from geniposidic acid and amino acids
end of the experiment. The LD50 values of these
aqueous pigments must therefore be greater than
50mlkgÿ1 body wt, that is, 88.8gkgÿ1 body wt on the
colour content of PRA (A = 500) and 276gkgÿ1 body
wt on that of PRG (A = 500), respectively.
In a 5-month oral toxicity evaluation, no group
showed toxic symptoms and no animal died from the
administration of arginine-red pigment. No treated
group of males or females showed signi®cant differ-
ences in body weight, food consumption or principal
internal organ weight from the control groups. No
histopathological changes were observed. Further, no
abnormality was detected by a microscopic examina-
tion of the organs removed.
These results showed that the maximum tolerable
dose of the arginine-red pigment of A = 1061 for the
mouse was more than 4.5% in the diet.
Mutagenicity tests of both pigments were negative,
including those for the highest dose, 5mg per plate.
CONCLUSIONRed pigments produced from geniposidic acid and
amino acids showed relatively high molecular masses,
heat stability, less changes in colour at pH 4±7 and low
toxicity. The electrostatic characters of the pigments
were governed by the amino acid used as a starting
material. It was suggested that this aqueous pigment is
available for coloration of foods.
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Table 1. Proportion of pigment filteredthrough the ultrafilter
Proportion of the pigment amount in ®ltrate to the test
solution (%)a
Filter unit PRA PRG
(Ultrafree-0.5, Millipore) in H2O in guanidine HCl in H2O in guanidine HCl
Biomax-10 (NMWLb 10,000) 10 41 2 24
Biomax-50 (NMWLb 50,000) 14 55 5 25
Biomax-100 (NMWLb 100,000) 33 88 28 51
a The proportion of the pigment ®ltered through each ®lter was calculated from the ratio of the absorbence of the
combined ultra®ltrates with that of the test solution.
A 0.5ml of the pigment solution of A=5 at lmax was centrifuged at 7740�g for 15min. The membrane of the
device was washed by spinning with 0.4ml of the same solvent at 7740�g for 30min. The ®ltrates were com-
bined and diluted to 1ml with the same solventb Nominal molecular weight limit.
814 J Sci Food Agric 79:810±814 (1999)
N Moritome, Y Kishi, S Fujii