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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Iridoglucosides from Gardenia jasminoides:Gardenoside and

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German).

2 Inouye H, Saito S and Shingu T, Shanzhiside, a new iridoid

glucoside from Gardenia jasminoides. Tetrahedron Letters

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3 Inouye H, Takeda Y, Saito H, Nishimura H and Sakuragi R,

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Zasshi 94:577±586 (1974) (in Japanese). CA 81:49977k.

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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