Neocrocin A: A novel crocetin glycoside with a unique system for binding sugars isolated from gardenia yellow

Article abstract of DOI:10.1248/cpb.55.1643

A novel crocetin glycosyl ester, neocrocin A (2), was isolated from gardenia yellow. The structure of 2 was elucidated as that of an all-trans-crocetin β-D-gentiobiosyl β-D-glucopyranosyl-(1→6)-D-2- deoxy-glucopyranos-2-yl diester based on chemical and spectral data. The findings provide evidence that the binding system of crocetin glycosides is not limited to the anomeric position.

Full text of DOI:10.1248/cpb.55.1643

November 2007
Notes
Chem. Pharm. Bull. 55(11) 1643—1646 (2007)
1643
Neocrocin A: a Novel Crocetin Glycoside with a Unique System for
Binding Sugars Isolated from Gardenia Yellow
Yoshinori UEKUSA,a,1) Naoki SUGIMOTO, ,b Kyoko SATO,b Young Sook YUN,a Akira KUNUGI,a
*
b
Takeshi YAMAZAKI,b and Ken-ichi TANAMOTO
a School of Life Science, Tokyo University of Pharmacy and Life Science; 1432–1 Horinouchi, Hachioji, Tokyo 192–0392,
Japan: and b National Institute of Health Sciences; 1–18–1 Kamiyoga, Setagaya-ku, Tokyo 158–8501, Japan.
Received June 20, 2007; accepted August 29, 2007; published online September 3, 2007
A novel crocetin glycosyl ester, neocrocin A (2), was isolated from gardenia yellow. The structure of 2 was
elucidated as that of an all-trans-crocetin b-D-gentiobiosyl b-D-glucopyranosyl-(1→6)-D-2-deoxy-glucopyranos-2-
yl diester based on chemical and spectral data. The findings provide evidence that the binding system of crocetin
glycosides is not limited to the anomeric position.
Key words Gardenia jasminoides; gardenia yellow; neocrocin A; crocetin glycoside; crocin
Crocin (1)2) is a digentiobiosyl all-trans-crocetin (8,8ꢀ-di- tained, respectively, after the methanolysis and hydrolysis7) of
apocarotene-8,8ꢀ-dioic acid) ester that is the major yellow 2. These observations implied that 2 had the same carotenoid
pigment in gardenia yellow and saffron, which are extracts of moiety, all-trans-crocetin, as the chromophore group, but
Gardenia jasminoides fruits and Crocus sativus stigmas, re- that the binding system for glucoses differed from that of 1.
1
spectively.3,4) Gardenia yellow and saffron consist of many The H- and 13C-NMR spectra of 1 showed simple subduple
minor pigments as well as some relatively abundant pigments signals, because 1 had C2 structural symmetry. The 1H-NMR
which have been characterized as all-trans- and 13-cis-cro- spectra of 2 were similar to, but more complex than, those of
cetin monoglucosyl ester,2) diglucosyl ester,2) monogentio- 1. Based on this comparison, we predicted that the C2 struc-
biosyl ester,2,5) glucosyl gentiobiosyl ester2) and gentiobiosyl tural symmetry was disrupted in 2 by the different binding
neapolitanosyl ester.5) However, the structures of the other system for glucoses at each end of crocetin, and that equili-
minor pigments have so far remained uncertain, and the brated isomerization could occur readily in the NMR solvent.
1
binding systems for sugars have not previously been con- The H-NMR spectrum of 2 revealed a crocetin moiety (dH
firmed by detailed spectroscopic investigations, such as 6.49—7.31), anomeric doublets of b configuration (dH 5.38
NMR analysis, after isolation. Here we report on the isola- (d, Jꢂ7.8 Hz), 4.53 (d, Jꢂ6.9 Hz), 4.13 (d, Jꢂ7.8 Hz), 4.15
tion and structural elucidation of a novel crocetin glycoside, (d, Jꢂ7.8 Hz)) and an anomeric doublet of a configuration
neocrocin A (2), which has a unique binding system for sug- (dH 5.06 (d, Jꢂ3.6 Hz)). Two anomeric protons (dH 4.53,
ars, based on spectral data and chemical derivatization (Fig. 5.06) were shifted to a high magnetic field, and two oxyme-
1).
thines on H-2 (dH 4.39 (dd, Jꢂ3.7, 10.1 Hz), 4.53 (t,
Jꢂ6.9 Hz)) were shifted to a low magnetic field, in compari-
son to those of 1. This observation indicated the existence of
Results and Discussion
Gardenia yellow extracted from dried gardenia fruits was a free hydroxyl group at an anomeric position on the glyco-
fractionated on a Diaion HP-20 column. The 60—70% syl groups. Furthermore, HMBC correlations were observed
methanol eluate was then concentrated and the residue was between the H-2 of the a-glucoside and b-glucoside (glucose
loaded into a preparative LC-MS system,6) led to the isola- C) at dH 5.06 and dH 4.53, and the carbonyl carbons on the
tion of crocin (1) and neocrocin A (2).
crocetin moiety at dC 167.92 and dC 167.11, respectively.
Neocrocin A (2) was isolated as a red amorphous powder. Based on these chemical and spectral data, 2 was determined
The molecular formula of 2 was established as C44H64O24, to be an all-trans-crocetin b-D-gentiobiosyl b-D-glucopyra-
→
which was as the same as that of 1, according to HR-ESI-MS nosyl-(1 6)-D-2-deoxy-glucopyranos-2-yl ester. The NMR
(m/z 999.3707, [MꢁNa]ꢁ, Calcd 999.3685), and the IR spec- spectral data for 1 and 2 are summarized in Table 1.
trum and UV/Vis absorption were similar to those of 1. All-
Furthermore, to confirm the binding system for the sugars
trans-crocetin dimethyl ester (3) and D-glucose were ob- of neocrocin A (2), we firstly carried out peraceylation of 2.
However, the sufficient quantity of peracetylated 2 was not
obtained for the structure determination, because 2 was un-
stable more than crocin (1). Hence, after peracetylation of
gardenia yellow, we isolated peracetylated crocin (1a) and an
isomer of neocrocin A (2a). The other isomer of peracety-
lated 2 could not be isolated using preparative LC-MS be-
cause of overlapping with the peak of 1a. The HR-ESI-MS
spectra indicated that the molecular formula of both 1a and
1
2a was C72H92O38. To compare the H- and 13C-NMR data
between 2a and 1a, detailed 2D-NMR experiments were per-
formed and the coupling constants were assigned using 1D-
Fig. 1. Chemical Structures of Crocin (1) and Neocrocin A (2)
TOCSY. The chemical shifts and coupling constants of glu-
∗ To whom correspondence should be addressed. e-mail: nsugimot@nihs.go.jp
© 2007 Pharmaceutical Society of Japan

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Vol. 55, No. 11
d
d
b
d
d
d
d
a
d
d
d
d

November 2007
1645
Table 2. 1H (800 Mz)- and 13C (200 MHz)-NMR Spectral Dataa) of Peracetylated Crocin (1a) and Neocrocin A (2a) (in CDCl3)
Peracetylated crocin (1a)b)
Peracetylated neocrocin A (2a) (a isomer)b)
No.
dH
dC
No.
dH
dC
No.
dH
dC
8, 8ꢀ
9, 9ꢀ
166.09
124.57
141.45
123.66
145.42
138.00
136.95
131.83
12.87
8
9
166.07
124.55
141.45
123.71
145.42
136.96
136.33
131.84
12.70
8ꢀ
9ꢀ
166.92
125.02
140.46
123.63
144.85
136.85
136.02
131.67
12.80
10, 10ꢀ 7.33 (d, 12.4)
11, 11ꢀ 6.54 (dd, 11.7, 14.9)
12, 12ꢀ 6.68 (d, 14.5)
13, 13ꢀ
14, 14ꢀ 6.42 (d, 10.1)
15, 15ꢀ 6.73 (dd, 2.1, 8.0)
19, 19ꢀ 1.98 (s)
10
11
12
13
14
15
19
20
7.32 (d, 11.4)
6.53 (dd, 11.5, 15.1)
6.68 (d, 15.2)
10ꢀ 7.20 (d, 12.4)
11ꢀ 6.50 (dd, 11.7, 14.9)
12ꢀ 6.60 (d, 15.1)
13ꢀ
14ꢀ 6.39 (d, 10.1)
15ꢀ 6.72 (br dd)
19ꢀ 1.91 (s)
6.41 (d, 9.7)
6.71 (br dd)
1.97 (s)
20, 20ꢀ 1.99 (s)
12.73
1.98 (s)
12.90
20ꢀ 1.98 (s)
12.90
Glucose A, C
Glucose A
Glucose C
1
2
3
4
5
6
5.74 (d, 8.2)
5.20 (t, 9.0)
5.27 (t, 9.4)
5.01 (t, 9.6)
3.83 (ddd, 2.4, 6.4, 10.5)
3.59 (dd, 5.8, 11.7)
3.93 (dd, 2.3, 11.4)
92.08
70.29
72.90
68.42
74.01
67.68
1
2
3
4
5
6
5.74 (d, 7.8)
5.20 (t, 9.0)
5.26 (t, 9.6)
5.01 (t, 9.8)
3.82 (m)
3.59 (dd, 5.8, 11.7)
3.92 (1H, br d)
92.08
70.29
72.90
68.38
74.01
67.68
1
2
3
4
5
6
6.73 (d, 3.7)
5.07 (dd, 3.9, 10.3)
5.55 (t, 9.9)
5.03 (t, 10.1)
4.05 (m)
3.53 (dd, 6.0, 11.0)
3.93 (br d)
88.99
69.63
70.08
68.47
70.99
67.96
Glucose B, D
Glucose B
Glucose D
1
2
3
4
5
6
4.55 (d, 8.2)
4.98 (dd, 7.8, 9.6)
5.17 (t, 9.4)
100.65
70.99
72.90
68.42
71.98
61.88
1
2
3
4
5
6
4.55 (d, 8.2)
4.97 (t, 8.9)
5.17 (t, 9.4)
5.05 (t, 9.4)
100.65
70.99
72.90
68.43
71.98
61.88
1
2
3
4
5
6
4.53 (d, 7.8)
4.97 (t, 8.9)
5.19 (t, 9.4)
5.07 (t, 9.9)
3.67 (ddd, 2.4, 4.5, 10.0)
4.11 (dd, 2.1, 12.2)
4.26 (dd, 4.6, 12.4)
100.85
70.99
72.76
69.03
72.02
61.88
5.05 (t, 10.2)
3.63 (ddd, 2.3, 4.6, 10.1)
4.09 (dd, 2.3, 12.4)
4.24 (dd, 4.6, 12.4)
3.63 (m)
4.09 (dd, 1.8, 12.4)
4.23 (dd, 5.0, 12.3)
1.98—2.06 (s)
Acetyl 2.00—2.07 (s)
20.65—20.80
20.65—20.94
169.45—170.73
169.36—170.79
a) Chemical shifts are in ppm and coupling constants in Hz are in parentheses. b) Assignments were made using 1D-TOCSY, COSY, HMQC and HMBC.
cose A and B of 2a were identical to those of 1a, and the
HMBC correlations of the H-1 (dH 5.74) of glucose A to C-8
(dC 166.07) of crocetin, and the H-1 (dH 4.55) of glucose B
to C-6 (dC 67.68) of glucose A, suggested the existence of a
gentiobiosyl moiety connected to crocetin at the anomeric
position, similar to in 1a. By contrast, the chemical shifts and
coupling constants of glucose C suggested that the anomeric
position of glucose C was in the a configuration, and the
HMBC correlations of the H-2 (dH 5.07) of glucose C to C-
8ꢀ (dC 166.07) of crocetin, and the H-1 (dH 4.53) of glucose
D to C-6 (dC 67.96) of glucose C, supported the notion that
another gentiobiosyl moiety was connected to the crocetin
1
via the number 2 position on glucose C. The H- and 13C-
NMR data for 1a and 2a are summarized in Table 2 and key
correlations of 2a are shown in Fig. 2. These observations
provided evidence that neocrocin A (2) possessed a unique
binding system for sugars.
Fig. 2. Key Correlations of Peracetylated Neocrocin A (2a)
As neocrocin A (2) isolated from gardenia yellow was a
mixture of isomers along with crocin (1), we initially sus- in nature. We are further investigating the possibility that
pected that neocrocin A (2) was an artefact formed by crocin other types of crocetin glycoside might also exist in this fruit
(1) via the rearrangement of the acyl moiety during the isola- through an ongoing study.
tion process. However, the LC profile of gardenia yellow,
Experimental
especially the peak height of neocrocin A (2), remained
unchanged after dissolving again with ethanol and water
under acidic and basic conditions. We thus concluded
that neocrocin A (2) was an original constituent of the G.
jasminoides fruits. To our knowledge, this is the first pub-
lished evidence that the binding system for the sugars of a
General IR spectra were recorded on a JASCO FT/IR-4100. UV/Vis
spectra were recorded on a JASCO UV-560. FAB-MS spectra were meas-
ured with JEOL JMS-700 system and ESI-MS spectra were measured with
JEOL JMS-T100LC “AccuTOF”. NMR spectra were recorded on JEOL
JNM-ECA (800 MHz) and JNM-ECA (500 MHz). Diaion HP-20 was used
for open-column chromatography. The preparative LC-MS system (Waters
FractionLynxTMMS auto purification system), which is described in our pre-
crocetin glycosyl ester is not limited to the anomeric position vious report,6) was used for isolation of compounds on this research. The

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Vol. 55, No. 11
solvents for LC-MS were purchased from Sigma-Aldrich Japan. All of the
other chemicals were of reagent or HPLC grade and were used without fur-
ther purification.
Plant Material The dried gardenia fruits, defined as the fruits of
Gardenia jasminoides, were purchased from Uchida Wakanyaku Co., Ltd.,
Japan, in July 2004. The crude drug name was Gardeniae Fructus (Serial
No.: VMAMQ), and the voucher specimen (GJ 72004) was deposited at Di-
vision of Food Additives in National Institute of Health Sciences.
1.98 (6H, s, H-20, 20ꢀ), 1.99 (6H, s, H-19, 19ꢀ), 3.75 (6H, s, MeOꢃ2), 6.35
(2H, br d, Jꢂ10.0 Hz, H-14, 14ꢀ), 6.53 (2H, dd, Jꢂ11.0, 15.0 Hz, H-11, 11ꢀ),
6.60 (2H, d, Jꢂ15.0 Hz, H-12, 12ꢀ), 6.70 (2H, dd, Jꢂ3.0, 8.0 Hz, H-15, 15ꢀ),
7.28 (2H, dd, Jꢂ1.4, 11.0 Hz, H-10, 10ꢀ); 13C-NMR (CDCl3, 125 MHz) d:
12.87 (C-20, 20ꢀ), 12.99 (C-19, 19ꢀ), 51.92 (MeOꢃ2), 123.88 (C-11, 11ꢀ),
126.48 (C-9, 9ꢀ), 131.40 (C-15, 15ꢀ), 135.43 (C-14, 14ꢀ), 136.79 (C-13, 13ꢀ),
138.97 (C-10, 10ꢀ), 143.84 (C-12, 12ꢀ), 169.02 (C-8, 8ꢀ).
Peracetylated Crocin (1a) and Neocrocine A (2a) Peracetylation of
gardenia yellow (1.0 g) was carried out with Ac2O/pyridine (cat. DMAP),
affording peracetylated crocin (1a, 1.5 g) (tR 26.0 min) and the a isomer of
neocrocin A (2a, 7.4 mg) (tR 27.9 min) using preparative LC/MS6) under the
following conditions: column, YMC J’sphere ODS-L80 (5 mm, 20 mm
i.d.ꢃ250 mm); mobile phase, H2O : MeCNꢂ42 : 58; flow rate, 10 ml/min;
make-up liquid, MeOH 1.0 ml/min; injection volume, 200 ml; detection and
collection trigger, ESI (positive mode), m/z 1587.5. The overall procedure
was repeated approximately 30 times. The b isomer of peracetylated 2 could
not be isolated because of overlapping with the peak of 1a.
Extraction and Isolation Gardenia yellow was extracted by stirring
dried gardenia fruits (1 kg) with 50% aqueous ethanol at ambient tempera-
ture in the dark for 2 h. The part of extracted gardenia yellow (2.25 g) was
subsequently fractionated on a Diaion HP-20 column (40 mm i.d.ꢃ250 mm)
by successive elution with 0—100% MeOH. The 60—70% methanol eluate
was then concentrated in vacuo and the residue was loaded into a preparative
LC-MS system.6) The conditions were as follows: column, Waters XTerra®
Prep MS C18 (5 mm, 19 mm i.d.ꢃ100 mm); mobile phase, H2O : MeOHꢂ
58 : 42; flow rate, 10 ml/min; make-up liquid, MeOH 1.0 ml/min; injection
volume, 200 ml; and detection and collection trigger, ESI (pos.) m/z 999.5.
The electrospray source ran at a 3.0 kV capillary voltage, with 120 and
350 °C source and desolvation temperatures, respectively, and with 350 and
60 l/h desolvation and cone gases, respectively. The cone voltage was 50 V.
Full-scan acquisition between m/z 100 and 2000 was performed at a scan
speed of 0.3 s/scan, with a 0.1-s inter-scan delay. The solvent delivered to the
electrospray interface was split in a 1 : 4 ratio, delivering around 200 ml/min
to the interface. The peaks of crocin (1) (retention time (tR) 15.3 min) and
neocrocin A (2) (tR 19.0 min) were each fractionated and concentrated in
vacuo at ꢄ40 °C. The overall procedure was repeated approximately 25
times to afford 220 mg of crocin (1) and 18 mg of neocrocin A (2).
1a red amorphous powder. IR (KBr) nmax cmꢅ1: 1063, 1222, 1371, 1615,
1751, 2944. UV/Vis (MeOH) lmax (log e): 436 (4.95), 462 (4.92), 260
(4.05): HR-ESI-MS m/z: 1587.5156 [MꢁNa]ꢁ (Calcd for C72H92O38Na:
1587.5164). 2a (an a isomer): red amorphous powder. IR (KBr) nmax cmꢅ1
:
1038, 1223, 1375, 1637, 1752, 2945. UV/Vis (MeOH) lmax (log e): 438
(4.81), 460 (4.77), 260 (4.05). HR-ESI-MS m/z 1587.5192 [MꢁNa]ꢁ (Calcd
for C72H92O38Na: 1587.5164); 1H (800 MHz)- and 13C (200 MHz)-NMR
spectral data of 1a and 2a: see Table 2.
References and Notes
1) Present address: School of Food and Nutritional Sciences, University
of Shizuoka; 52–1 Yada, Suruga, Shizuoka 422–8526, Japan.
2) Calsteren M. V., Bissonnette M. C., Cormier F., Dufresne C., Ichi T.,
LeBlanc J. C. Y., Perreault D., Roewer I., J. Agric. Food Chem., 45,
1055—1061 (1997).
Crocin (1)2) Red amorphous powder. IR (KBr) nmax cmꢅ1: 1069, 1227,
1271, 1577, 1699, 2920, 3398. UV/Vis (MeOH) lmax (log e): 433 (4.49),
458 (4.46), 259 (3.65). HR-ESI-MS m/z: 999.3674 [MꢁNa]ꢁ (Calcd for
1
C44H64O24Na: 999.3685). H (800 MHz)- and 13C (200 MHz)-NMR spectral
data: see Table 1.
3) Carmona M., Zalacain A., Sánchez A. M., Novella J. L., Alonso G. L.,
J. Agric. Food Chem., 54, 973—979 (2006).
4) Ichi T., Higashimura Y., Katayama T., Koda T., Shimizu T., Tada M. J.,
Jpn. Soc. Food Sci. Technol., 42, 776—783 (1995).
5) Pfister S., Meyer P., Steck A., Pfander H., J. Agric. Food Chem., 44,
2612—2615 (1996).
Neocrocin A (2) Red amorphous powder. IR (KBr) nmax cmꢅ1: 1023,
1227, 1270, 1578, 1702, 2918, 3375. UV/Vis (MeOH) lmax (log e): 432
(4.73), 457 (4.69), 258 (3.97). HR-ESI-MS m/z 999.3707 [MꢁNa]ꢁ (Calcd
for C44H64O24Na, 999.3685). 1H (800 MHz)- and 13C (200 MHz)-NMR spec-
tral data: see Table 1.
Crocetin Dimethyl Ester (3) Methanolysis of 2 (10 mg) with 5% HCl
methanol at room temperature for 50 h resulted in red amorphous powder (3,
3 mg) after purification by Silica gel column chromatography (EtOAc :
6) Sato K., Sugimoto N., Ohta M., Yamazaki T., Maitani T., Tanamoto K.,
Food Add. Contam., 20, 1015—1022 (2003).
7) Kawahara N., Tamura T., Inoue M., Hosoe T., Kawai K., Sekita S.,
Satake M., Goda Y., Phytochemistry, 65, 2577—2581 (2004).
1
hexaneꢂ2 : 3). ESI-MS m/z: 327 [MꢅH]ꢅ. H-NMR (CDCl3, 500 MHz) d: