Physical stability of the blue pigments formed from geniposide of gardenia fruits: Effects of ph, temperature, and light

Article abstract of DOI:10.1021/jf000978f

Fruits of Gardenia jasminoides contain geniposide which can be transformed to blue pigments by a simple modification. Colorless geniposide obtained from gardenia, fruits by charcoal and silica gel column chromatographies was hydrolyzed with β-glucosidase to yield genipin. The resulting genipin was transformed to blue pigments by reaction with amino acids (glycine, lysine, or phenylalanine). The stability of the blue pigments against heat, light, and pH was studied to examine the blue dye for possible use as' a value-added food colorant. Thermal degradation reactions at temperatures of 60-90 °C were carried out at different pH levels within the range 5.0-9.0 (pH 5.0, acetate buffer; pH 7.0, phosphate buffer; and pH 9.0, CHES buffer). The blue pigments remained Stable after 10 h at temperatures of 60-90 °c, and in some cases, more new pigments formed. The pigments were more stable at alkaline pH than neutral and acidic pH. Similarly, the pigments were stable under light irradiance of 5000-20 000 lux. In this case, pH effect was not significant.

Full text of DOI:10.1021/jf000978f

430
J. Agric. Food Chem. 2001, 49, 430−432
P h ysica l Sta bility of th e Blu e P igm en ts F or m ed fr om Gen ip osid e of
Ga r d en ia F r u its: Effects of p H, Tem p er a tu r e, a n d Ligh t
,
†
†
‡
‡
Young-Sook Paik,* Chang-Min Lee, Man-Ho Cho, and Tae-Ryong Hahn
Department of Chemistry and Genetic Engineering, and Plant Metabolism Research Center,
Kyung Hee University, Suwon 449-701, Korea
Fruits of Gardenia jasminoides contain geniposide which can be transformed to blue pigments by
a simple modification. Colorless geniposide obtained from gardenia fruits by charcoal and silica gel
column chromatographies was hydrolyzed with â-glucosidase to yield genipin. The resulting genipin
was transformed to blue pigments by reaction with amino acids (glycine, lysine, or phenylalanine).
The stability of the blue pigments against heat, light, and pH was studied to examine the blue dye
for possible use as a value-added food colorant. Thermal degradation reactions at temperatures of
6
0-90 °C were carried out at different pH levels within the range 5.0-9.0 (pH 5.0, acetate buffer;
pH 7.0, phosphate buffer; and pH 9.0, CHES buffer). The blue pigments remained stable after 10
h at temperatures of 60-90 °C, and in some cases, more new pigments formed. The pigments were
more stable at alkaline pH than neutral and acidic pH. Similarly, the pigments were stable under
light irradiance of 5000-20 000 lux. In this case, pH effect was not significant.
Keyw or d s: Gardenia jasminoides; geniposide; genipin; glycine; lysine; phenylalanine; physical
stability; gardenia blue pigments
INTRODUCTION
Fruits of Gardenia jasminoides, easily obtainable in
southern parts of the Korea peninsula, have long been
used as folklore medicine for antiphlogistic, diuretic,
laxative, choleretic, and hemostatic purposes in the
treatment of trauma by external application (1). Gar-
denia fruits also have been used as a yellow dye for
staining foods and fabrics. The yellow color components
of gardenia fruits are composed of carotenoids and
related compounds (1, 2). Colorless components of
gardenia fruits can also produce blue colorants by a
simple modification of an enzyme reaction followed by
the treatment of primary amines (2). With growing
concern over the safety of synthetic dyes, the importance
of natural colorants suitable for use in foods has gained
increasing attention. The only natural blue colorants
commercially feasible today are those derived from
gardenia fruits and algae (3). The protein dye phyco-
cyanin, derived from blue green algae, has stability
problems as a colorant in alcoholic beverages or in
aqueous solutions of different pH levels (3). The blue
pigments produced by gardenia fruits have been widely
used in east Asia, including Korea and J apan. Although
the effect of pH on gardenia blue pigments was reported
to be relatively stable (3), a detailed description of the
physical stability of the pigments has yet to be reported.
In this research we isolated geniposide 1 (Figure 1),
a colorless iridoid glycoside, from gardenia fruits by
charcoal and silica gel column chromatographies. Geni-
poside was hydrolyzed with â-glucosidase to produce
aglycon genipin 2 (Figure 1). The resulting genipin was
transformed to blue pigments by reactions with amino
F igu r e 1. Chemical structures of geniposide 1 and genipin 2
and the procedure of blue pigments formation from geniposide
of gardenia fruits.
acids (glycine, lysine, or phenylalanine). Thermal and
light stabilities of the blue pigments in aqueous solu-
tions were tested at pHs within the range of 5.0-9.0.
EXPERIMENTAL PROCEDURES
Ma ter ia ls. Dried fruits of Gardenia jasminoides were
obtained from the local region of Kyungnam province and
stored in the refrigerator. The silica gel for column chroma-
tography (Kiesel gel 230-400 mesh) and TLC plate (Kiesel
gel 60 F254) were purchased from Merck. â-Glucosidase, glycine,
lysine, phenylalanine, and reagents for buffer preparation,
including CHES (2-[N-cyclohexylamino]ethanesulfonic acid)
and sodium phosphate, were purchased from Sigma Chemical
Co. Other chemicals including chloroform, ethanol, and metha-
nol were obtained from Hayman Ltd. and Tedia Company, Inc.
*
To whom correspondence should be addressed. Tel: 82-
3
1-201-2422. Fax: 82-31-202-7337. E-mail: paikys@khu.ac.kr.
†
Department of Chemistry.
‡
Department of Genetic Engineering.
1
0.1021/jf000978f CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/20/2000

Physical Stability of Gardenia Blue Pigments
J. Agric. Food Chem., Vol. 49, No. 1, 2001 431
Sp ectr a l An a lysis. The UV-vis spectra were recorded on
in a phytotron (Vision Co.) with different light intensities of
5000-20 000 lux. Light intensities were adjusted with a lux
meter and sample cuvettes were placed on it. Thermal
degradation and photodegradation of the blue pigments were
monitored spectrophotometrically at 580 nm.
1
a Milton Roy Spectronics 3000 spectrophotometer. The
H
1
3
NMR and C NMR spectra were measured on a 400 MHz FT-
NMR(J EOL) at 400 and 100 MHz, respectively. Melting points
were taken in open capillaries in a Mel-Temp apparatus and
are uncorrected. EIMS spectra were obtained with a J EOL
J MS-AS505 WA mass spectrometer.
RESULTS AND DISCUSSION
Isola tion of Gen ip osid e 1 a n d Gen ip in 2. Geniposide of
G. jasminoides was isolated by the methods of Endo and
Taguchi (4) and Lee et al. (5) with minor modifications.
Gardenia fruits (100 g) were ground and extracted with 0.5 L
Geniposide 1 obtained from gardenia fruits was
hydrolyzed with â-glucosidase and the resulting genipin
2
was transformed to the blue pigments by the reactions
with amino acids. Our previous work (5) with blue
pigments formation indicated that the optimum pH for
pigment formation was 7.0 and the best choice of the
amino acids was glycine, lysine, or phenylalanine. UV/
vis spectra for the formation of the blue pigments in
phosphate buffer (pH 7.0) with scanning intervals of 5
min showed that λmax at 240 nm of genipin disappeared
rapidly when genipin was treated with amino acids, and
λmax at about 290 nm of intermediates peak started to
appear, and finally λmax at about 570-600 nm produced
blue pigment polymers (5). Blue pigment polymers
contained many blue components (3). Absorption maxima
of the blue pigments formed from genipin with glycine,
lysine, and phenylalanine were 580, 583, and 589 nm,
respectively.
The stability of pigments is affected by many factors
such as heat, light, and pH (8). Thermal degradation
reactions of gardenia blue pigments were carried out
at pH of 5.0 (100 mM acetate buffer), 7.0 (100 mM
phosphate buffer), and 9.0 (100 mM CHES buffer) at
different temperatures within the range of 60-90 °C.
The degree of degradation was determined by measur-
ing absorbance changes at 580 nm. When the degree of
degradation was plotted on a semilogarithmic scale, the
plots showed a curved line, indicating that the reaction
did not follow simple first-order kinetics. Table 1 sum-
marizes thermal stabilities of the blue pigments formed
from genipin with glycine, lysine, and phenylalanine at
pH levels of 5.0, 7.0, and 9.0 and at temperatures of 60,
70, 80, and 90 °C. As shown in Table 1, the stability of
the blue pigments formed from genipin with glycine was
more stable in the alkaline condition than in acidic or
neutral conditions. For example, the percents of remain-
ing blue pigments after 10 h at 60 °C under the
conditions of pH 5.0, 7.0, and 9.0 were 97%, 99%, and
of CHCl
3
three times to remove lipid components. The dried
OH three times at
residue was extracted with 0.5 L of CH
3
room temperature for 3 h with stirring. The combined extracts
were concentrated to a small volume, dissolved into water, and
applied to charcoal. The concentrates in charcoal were eluted
with water, followed by 10% aqueous ethanol, and finally with
methanol. The methanol fractions were applied to a silica gel
column and eluted with a CHCl
then crystallized in acetone to give geniposide (1.95 g). Mp,
62-164 °C (163-164 °C, (4)), UV (CH OH) λmax 237 nm,
EIMS, m/z 388. H NMR (400 MHz, DMSO-d ) δ: 7.45 (s, H-3),
3 3
/CH OH mixture (7:3) and
1
3
1
6
5
.67 (br. s, H-7), 5.11 (d, J ) 6.8 Hz, H-1), 5.02 (d, J ) 5.4 Hz,
G2-OH), 4.96 (d, J ) 5.1 Hz, G3-OH), 4.92 (d, J ) 5.1 Hz, G4-
OH), 4.72 (t, J ) 5.4 Hz, 10-OH), 4.52 (d, J ) 7.8 Hz, H-G1),
4
3
.45 (t, J ) 5.8 Hz, G6-OH), 4.12 (br. d, J ) 15.0 Hz, H-10),
.96 (br. d, J ) 15.0 Hz, H-10), 3.64 (m, H-G6), 3.63 (s,
OCH ), 3.41 (m, H-G6), 3.16 (m, H-G3), 3.11 (m, H-G5), 3.05
3
-
(
m, H-G4), 3.05 (m, H-5), 2.97 (m, H-G2), 2.67 (m, H-6), 2.63
1
3
(m, H-9), 2.03 (m, H-6). C NMR (100 MHz, DMSO-d
6
) δ:
-), 151.6 (C-3), 144.1 (C-8), 125.5 (C-7), 110.9 (C-
), 98.6 (C-G1), 95.7 (C-1), 77.2 (C-G5), 76.6 (C-G3), 73.3 (C-
), 45.9
1
4
66.9 (-CO
2
G2), 70.0 (C-G4), 61.0 (C-G6), 59.3 (C-10), 51.0 (-OCH
3
(C-9), 38.0 (C-6), 34.4 (C-5).
Geniposide (1.0 g) and â-glucosidase (5 mg) were added into
0 mL of acetate buffer (pH 5.0) at 37 °C and stirred for 5 h.
5
The resulting aglycon was extracted with ether three times.
The combined extracts were treated with sodium sulfate
followed by filtration and concentration in vacuo. The concen-
trates were crystallized in ether to yield 0.5 g of genipin (4-
7
). Mp, 118-120 °C (120-121 °C, (7)), UV (CH
3
OH) λmax 240
) δ: 7.53 (s, H-3), 5.88 (s,
H-7), 4.82 (d, J ) 8.5 Hz, H-1), 4.35 (d, J ) 13.2 Hz, H-10),
1
nm, EIMS, m/z 226. H NMR (CDCl
3
4
9
2
1
1
6
.29 (d, J ) 13.2 Hz, H-10), 3.74 (s, -OCH ), 3.22 (ddd, J )
3
.5, 8.5, 8.5 Hz, H-5), 2.89 (ddt, J ) 16.8, 8.5, 1.4 Hz, H-6),
.54 (ddd, J ) 8.5, 8.5, 1.5 Hz, H-9), 2.07 (ddt, J ) 16.8, 9.5,
1
3
.8 Hz, H-6). C NMR (100 MHz, CDCl
3
) δ: 167.9 (-CO -),
2
52.4 (C-3), 142.1 (C-8), 130.9 (C-7), 110.8 (C-4), 96.3 (C-1),
1.3 (C-10), 51.3 (-OCH ), 48.2 (C-9), 39.0 (C-6), 36.7 (C-5).
F or m a t ion of Gen ip in t o Ga r d en ia Blu e P igm en t s.
3
1
05%, respectively. It seems likely that more blue
Genipin (10 mg) and amino acids (glycine, lysine, or phenyl-
alanine, 0.44 mmol) were added respectively into 2 mL of 100
mM phosphate buffer (pH 7.0) at 70 °C and stirred for 5 h.
The three reaction vessels containing blue pigments were
wrapped with aluminum foil and stored in the refrigerator for
further use.
pigments could be formed from monomer or dimer
intermediates at alkaline pH. Similar, but more dra-
matic, results occurred in the case of the blue pigments
formed from genipin and lysine. The percent of remain-
ing blue pigments after 10 h at 60 °C under the
conditions of pH 5.0, 7.0, and 9.0 were 104%, 102%, and
Effects of p H, Tem p er a tu r e, a n d Ligh t on Ga r d en ia
Blu e P igm en ts. Aliquots of the refrigerated blue pigments
1
10%, respectively. It is likely that the extra primary
(10 µl) were added into 0.99 mL of buffer solutions (pH 5.0,
amino group of lysine plays a crucial role for the
formation of the blue pigments. The percent of remain-
ing blue pigments formed from genipin and phenylala-
nine after 10 h at 60 °C under the conditions of pH 5.0,
1
9
00 mM acetate buffer; pH 7.0, 100 mM phosphate buffer; pH
.0, 100 mM CHES buffer). Thermal stabilities of the blue
pigments in the pH range of 5.0-9.0 were measured with
scanning intervals of 30 min at temperatures of 60-90 °C.
Light stabilities of the blue pigments at pHs within the range
of 5.0-9.0 were also tested at light intensities of 5000-20 000
lux (light source, metal halide lamp).
7
.0, and 9.0 were 95%, 99%, and 100%, respectively,
indicating that these blue pigments were also heat
stable components.
Sa m p le P r ep a r a tion a n d Rea ction Con d ition . Blue
pigments transformed from genipin and an amino acid were
dissolved in buffer solution. The pigment concentration was
adjusted to give an initial absorbance between 0.7 and 0.8 at
Fujikawa et al. (3) reported that the ratios of the
remaining absorbances of the gardenia blue pigments
after two weeks at 40 °C in the dark under different
pHs (pH 3-8) were 65-80%. These values are similar
to the present data even though reaction conditions are
different. Blue color of phycocyanin (3), obtained from
blue green algae, disappeared completely after two
weeks at 40 °C in the dark at pH 3, 4, and 8 in aqueous
λvis,max. Degradation reactions were carried out in 1.0-mL
quartz spectrophotometer cuvettes. The prepared samples
were degraded at 60, 70, 80, and 90 °C. Duplicate tests of
thermal degradation reactions were performed. Photodegra-
dation reactions of the blue pigments at 4 °C were carried out

432 J. Agric. Food Chem., Vol. 49, No. 1, 2001
Paik et al.
Ta ble 1. P er cen t Rem a in in g Blu e P igm en ts (A580) a fter Differ en t Tim es a t Va r iou s p H Levels a n d Tem p er a tu r es
pH 5.0 pH 7.0 pH 9.0
80 80 80
6
0
70
90 °C
60
70
90 °C
60
70
90°C
Gly
Lys
Phe
0 h
100
99
98
97
97
97
100
94
92
92
92
90
100
90
87
84
83
81
100
89
85
82
80
78
100
101
101
101
101
99
100
99
98
97
96
94
100
98
97
94
94
94
100
96
93
90
88
85
100
102
103
104
104
105
100
102
103
104
103
103
100
102
103
102
99
100
101
101
98
2
4
6
8
h
h
h
h
94
1
1
1
0 h
99
93
0 h
100
101
102
102
103
104
100
99
99
99
100
100
100
100
98
100
87
83
81
79
79
100
102
101
101
101
102
100
101
101
102
102
101
100
99
98
97
95
94
100
98
94
91
89
85
100
103
105
107
109
110
100
103
106
108
109
108
100
105
105
105
103
102
100
105
105
103
101
102
2
4
6
8
h
h
h
h
98
95
0 h
95
0 h
100
98
97
96
95
95
100
94
92
92
92
88
100
91
88
86
83
83
100
89
85
83
79
79
100
100
100
99
99
99
100
99
99
98
98
96
100
97
96
95
92
92
100
96
95
91
90
87
100
100
100
99
99
100
100
99
98
98
99
97
100
99
100
98
100
98
98
96
2
4
6
8
h
h
h
h
100
102
99
0 h
99
Ta ble 2. P er cen t Rem a in in g Blu e P igm en ts (A580) a fter Differ en t Tim es a t Va r iou s p H Levels a n d Ligh t In ten sities
pH 5.0
10 000
pH 7.0
10 000
pH 9.0
10 000
0
5000
20 000 lux
0
5000
20 000 lux
0
5000
20 000 lux
Gly
Lys
Phe
0 h
100
100
99
100
96
100
96
100
94
100
100
99
100
97
100
97
100
95
100
100
100
100
100
100
98
100
97
100
95
1
2
5
h
h
h
95
95
91
97
95
92
97
95
92
99
93
90
91
86
84
76
99
94
91
92
86
85
76
95
90
90
83
82
70
1
1
1
0 h
98
98
0 h
100
99
100
96
100
96
100
95
100
100
99
99
97
100
97
100
96
100
96
100
99
100
95
100
95
100
93
1
2
5
h
h
h
98
95
95
92
95
94
93
98
94
92
89
97
93
91
86
94
91
87
97
90
87
80
0 h
96
91
87
78
91
87
80
96
87
79
67
0 h
100
99
100
95
100
91
100
90
100
99
100
98
100
96
100
91
100
99
100
96
100
94
100
91
1
2
5
h
h
h
98
93
87
84
99
96
92
85
99
95
91
85
97
91
79
69
98
95
86
71
98
89
83
69
0 h
96
83
69
53
97
85
74
56
96
79
70
51
solutions, showing that the gardenia blue pigments have
much better stabilities than phycocyanin for use as
value-added food colorants.
LITERATURE CITED
(1) Tang, W.; Eisenbrand, G. Chinese Drugs of Plant Origin;
Springer-Verlag: Berlin, 1992; pp 539-543.
(
(
(
2) Hendry, G. A. F.; Houghton, J . D. Natural Food Colo-
rants, 2nd ed.; Chapman & Hall: London, 1996; pp
318-321.
The photodegradation reactions of blue pigments were
carried out under different light intensities of 5000-
3) Fujikawa, S.; Fukui, Y.; Koga, K.; Kumada, J . Brilliant
Skyblue Pigment Formation from Gardenia Fruits. J .
Ferment. Technol. 1987, 65, 419-424.
4) Endo, T.; Taguchi, H. The Constituents of Gardenia
jasminoides: Geniposide and Genipin-gentiobioside.
Chem. Pharm. Bull. 1973, 21, 2684-2688.
2
0 000 lux at pH 5.0, 7.0, and 9.0. These reactions were
carried out at 4 °C to study the effect of light for usage
in cold beverages and ices. Table 2 summarizes light
stability of the blue pigments (formed from genipin with
glycine, lysine, or phenylalanine) with and without light
for 10 h in solutions of pH 5.0, 7.0, and 9.0. As
summarized in Table 2, pH effect was not significant.
For example, the percent remaining absorbances of the
blue pigments from genipin with glycine after 10 h with
light intensity of 5000 lux under the conditions of pH
(5) Lee, J .-Y.; Hahn, T.-R.; Paik, Y.-S. Physicochemical
Characteristics for the Transformation of Blue Pigments
from Genipin of Gardenia jasminoides with Amino
Acids. Agric. Chem. Biotechnol. 1998, 41, 399-404.
(
6) Drewes, S. E.; Kayonga, L. Iridoid Molluscicidal Com-
pounds from Apodytes dimidiata. J . Nat. Prod. 1996,
59, 1169-1170.
5
.0, 7.0, and 9.0 were 90%, 91%, and 90%, respectively.
(
7) Djerassi, C.; Nakano, T.; J ames, A. N.; Zalkow, L. H.;
Eisenbraun, E. J .; Shoolery, J . N. Terpenoids XLVII. The
Structure of Genipin. J . Org. Chem. 1961, 26, 1192-
The percent remaining absorbances of the blue pigments
from genipin with glycine after 10 h with light intensity
of 20 000 lux under the conditions of pH 5.0, 7.0, and
1
206.
9
.0 were 76%, 76%, 70%, respectively. The blue pig-
(
8) Attoe, E. L.; von Elbe, J . H. Photochemical Degradation
of Betanine and Selected Anthocyanines. J . Food Sci.
1981, 46, 1934-1937.
ments formed from phenylalanine with the light inten-
sity of 20 000 lux at pH 9.0 after 10 h showed 51% of
remaining absorbance, indicating that this blue dye was
the least stable pigment when exposed to intense light.
In conclusion, gardenia blue pigments were very stable
with regard to pH, temperature, and light conditions
and may have potential for use as value-added colorants
for foods.
Received for review August 8, 2000. Revised manuscript
received October 23, 2000. Accepted October 30, 2000. This
work was supported by a grant from the National Science and
Engineering Foundation (2000) through the Plant Metabolism
Research Center.
J F000978F