C-prolinylquercetins from the yellow cocoon shell of the silkworm, Bombyx mori

Article abstract of DOI:10.1016/j.phytochem.2005.11.030

Two flavonoids containing the L-proline moiety, 6-C-[(2S,5S)-prolin-5-yl] quercetin (prolinalin A) and 6-C-[(2S,5R)-prolin-5-yl] quercetin (prolinalin B), were isolated from the cocoon shell of the silkworm, Bombyx mori. Their structural elucidation was achieved by application of acid hydrolysis and spectroscopic methods. These compounds were not found in the leaves of mulberry (Morus alba L.), the host plant of the silkworm, suggesting that the flavonoids are metabolites of the insect. This is the first time that flavonoids with an amino acid moiety have been found as naturally occurring compounds.

Full text of DOI:10.1016/j.phytochem.2005.11.030

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 579–583
www.elsevier.com/locate/phytochem
C-prolinylquercetins from the yellow cocoon
shell of the silkworm, Bombyx mori
a,
b
a
a
Chikara Hirayama *, Hiroshi Ono , Yasumori Tamura , Masatoshi Nakamura
a
National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan
b
National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan
Received 6 September 2005; accepted 26 November 2005
Available online 23 January 2006
Abstract
Two flavonoids containing the L-proline moiety, 6-C-[(2S,5S)-prolin-5-yl] quercetin (prolinalin A) and 6-C-[(2S,5R)-prolin-5-yl] quer-
cetin (prolinalin B), were isolated from the cocoon shell of the silkworm, Bombyx mori. Their structural elucidation was achieved by
application of acid hydrolysis and spectroscopic methods. These compounds were not found in the leaves of mulberry (Morus alba
L.), the host plant of the silkworm, suggesting that the flavonoids are metabolites of the insect. This is the first time that flavonoids with
an amino acid moiety have been found as naturally occurring compounds.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Silkworm; Bombyx mori; Mulberry; Morus alba; Moraceae; Flavonoid; Quercetin; L-Proline
1. Introduction
continuing search among silkworm strains for novel flavo-
noids with possible biological activity, we found that the
aqueous MeOH extract of the yellow cocoon shell of a Chi-
nese race of Daizo contain novel flavonoids with an amino
acid moiety. In the present study, we describe the isolation
and structural elucidation of two new compounds, desig-
nated as prolinalin A (1) and prolinalin B (2).
It has been reported that cocoon shells of some strains
of the silkworm, Bombyx mori, contain flavonoid-like pig-
ments (Oku, 1934; Hayashiya et al., 1959). Recently, Tam-
ura et al. (2002) isolated quercetin 5-glucoside, quercetin
5,40-diglucoside, and quercetin 5,7,40-triglucoside from
the yellow-green cocoon shell of a race Multi-Bi. These
flavonoids were not found to be present in leaves of its host
plant, mulberry tree (Morus alba), in which quercetin-3-
glycosides (rutin, isoquercitrin) and quercetin aglycon are
naturally occurring (Zhishen et al., 1999). This suggests
that flavonoids from the diet are modified within the silk-
worm by a glucosyltransferase that can transfer a glucose
residue to the C-5 hydroxy position of quercetin. Such an
enzyme has not been reported in animal tissues. Flavonoids
modified by B. mori may be useful as medicinal or cosmetic
materials because the ethanolic extracts of yellow-green
colored cocoon shells of a range of strains of B. mori have
potent antibacterial activity (Kurioka et al., 1999) and
strong antioxidant activity (Yamazaki et al., 1999). In a
OH
OH
O
O
HO
H
N
OH
HOOC
OH
1
OH
OH
O
HO
H
N
OH
HOOC
OH
O
*
Corresponding author. Tel.: +81 29 838 6087; fax: +81 29 838 6028.
2
E-mail address: hirayam@nias.affrc.go.jp (C. Hirayama).
0031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.11.030

580
C. Hirayama et al. / Phytochemistry 67 (2006) 579–583
2. Results and discussion
8.5 Hz), d 7.62 (1H, dd, J = 2.1 and 8.5 Hz), and d 7.73
(1H, d, J = 2.1 Hz), which were assigned to H-50, H-60,
and H-20, respectively. A one-proton singlet at d 6.45 sug-
gested the presence of a substitution at C-6 or C-8. The
13C NMR spectrum displayed 15 distinct carbon signals,
which were assigned to the quercetin moiety, including a
carbonyl carbon (d 177.3) and five oxygenated aromatic
carbons (d 146.3, 149.0, 157.9, 160.2, 165.1). The absence
of a methine carbon signal in ring-A, but the presence of
a quaternary carbon signal at d 105.3, indicated a ring-A
substitution.
The aqueous MeOH extract of the cocoon shell of the
silkworm, B. mori, was purified as described in the experi-
mental section to yield two new compounds. Prolinalin A
(1) was obtained as a yellow amorphous solid. The UV
absorption data of the compound in MeOH were similar
to those of quercetin. The UV spectral changes with various
shift reagents were indicative of the presence of a flavonol
skeleton with free hydroxyl groups at C-3, C-5, C-7, and
C-40 positions, and the presence of a free ortho-dihydroxyl
group (Markham, 1982). The compound gave a red-purple
pigment by reaction with ninhydrin reagent, indicating that
it contains nitrogen. The compound was resistant to hydro-
lysis by 0.1 N HCl at 100 ꢁC, but afforded d-pyroline-5-car-
boxylic acid in 6 N HCl at 110 ꢁC. The D/L configuration of
d-pyroline-5-carboxylic acid was determined to be L by the
chemical correlation to L-proline. The high-resolution FT-
ICR-MS displayed a protonated ion at m/z 416 (observed
416.0973, calculated 416.0976 for C20H18NO9), and prod-
uct ions from [M + H]+ were observed at m/z 303 and 370
by MS/MS experiments. From these results, we considered
1 to be a quercetin derivative with an L-proline moiety.
1
Residual H and 13C resonances of 1 were observed in
the relatively up-field region of the spectra. The DQF-
COSY, HSQC, and HMBC spectra showed that the resid-
ual moiety contained the fragment –CO–CH–CH2–CH2–
CH–. Signals at d4.30 (dd, J = 7.7, 9.6) and d 5.27 (dd,
J = 7.3, 10.8) were assigned to H-200 and H-500, respectively.
Furthermore, the signals at d 2.12 (dddd, J = 7.1, 9.6, 11.1,
13.1) and d 2.62 (dddd, J = 2.1, 7.4, 7.7, 13.1) were attrib-
utable to H-300, while the signals at d 2.30 (dddd, J = 2.1,
7.1, 7.3, 12.8) and d 2.40 (dddd, J = 7.4, 10.8, 11.1, 12.8)
could be assigned to H-400. In addition, the five carbon res-
onances were assigned, respectively, to d 174.3 to the car-
boxyl carbon (C-600), and d 63.4, 31.6, 31.5, and 56.1 to
1
Finally, the structural elucidation was established by H
and 13C NMR spectroscopic analysis.
the alicyclic carbons (C-200, C-300, C-400, and C-500). The H
1
The 1H and 13C NMR spectroscopic data (Table 1)
were similar to those of quercetin (Markham and Geiger,
and 13 C NMR spectroscopic data for the moiety were con-
sistent with those of C-5-substituted proline derivatives
(Severino et al., 2003). These NMR observations and the
acidolysis results indicated the presence of an L-prolin-5-
yl substituent in 1.
1
1994; Markham et al., 1978). From the H NMR data, a
30,40-dihydroxy group in ring-B was evidenced by three
aromatic resonances located at d 6.88 (1H, d, J =
Table 1
13C NMR (125 MHz) and 1H NMR (500 MHz) Spectroscopic data for compounds 1 and 2 (d ppm, in CD3OD)
Position
1
2
d C
d H
d C
d H
2
3
4
5
6
148.3
137.4
177.3
160.2
105.3
165.1
94.9
147.8
137.1
177.0
160.1
104.3
165.0
95.7
7
8
6.45s
6.38s
9
157.9
103.9
124.0
116.1
146.3
149.0
116.3
121.7
63.4
158.3
103.0
124.2
116.0
146.3
148.8
116.3
121.7
61.8
10
10
20
30
40
500
600
2
7.73d (2.1)a
7.74br, s
6.88d (8.5)
7.62dd (2.1, 8.5)
4.30dd (7.7, 9.6)
a: 2.12dddd (7.1, 9.6, 11.1, 13.1)
b: 2.62dddd (2.1, 7.4, 7.7, 13.1)
6.88br, s
7.62br, s
4.08dd (5.1, 10.4)
a: 2.33m
b: 2.51m
300
31.6
30.0
400
31.5
a: 2.30dddd (2.1, 7.1, 7.3, 12.8)
b: 2.40dddd (7.4, 10.8, 11.1, 12.8)
29.6
a: 2.24m
b: 2.27m
500
600
a
56.1
174.3
5.27dd (7.3, 10.8)
57.2
174.6
5.05t (7.9)
J values in parentheses are recorded in Hz.

C. Hirayama et al. / Phytochemistry 67 (2006) 579–583
581
´
Comparison of the carbon shifts of 1 with those of pub-
lished data for quercetin (Markham et al., 1978) revealed a
down-field shift of C-6 (Dd 7.0 ppm ), which indicated that
the proline substitution occurred at C-6. The linkage of the
L-prolin-5-yl group at C-6 was also deduced from an
HMBC experiment, which showed correlations of the reso-
nance at d 5.27 (H-500) to carbon signals at d 105.3 (C-6),
160.2 (C-5), and 165.1 (C-7), while the resonance at d
6.45 (H-8) showed correlations to the signals at d 103.9
(C-10),d 157.9 (C-9) and d 165.1 (C-7) (Fig. 1). On the basis
of these observations, the structure 1 was determined as 6-
C-(prolin-5-yl) quercetin.
The stereochemistry of 1 was determined by the 1,2-diax-
ial relationships between H-200 and Ha-300 (J = 9.6), Ha-300
and Hb-400 (J = 11.1), and Hb-400 and H-500 (J = 10.8), as
shown in Fig. 1. These large coupling constants (9.6 ꢀ
11.1) suggested that H-200, Ha-300, Hb-400, and H-500, respec-
tively, occupy pseudo-axial positions, with respect to the
five-membered ring. This configuration should be observed
when the five-membered ring adopts an envelope conforma-
tion, in which C-200, C-400, C-500, and N atoms of the ring are
coplanar, with C-300 displaced above that plane. In this case,
the carboxyl group at C-200 and the quercetin at C-500 should
be trans on the pyrrolidine ring. Based on the L-configura-
tion of the d-pyroline-5-carboxylic acid, acidolysis product
of 1, the absolute configuration of pyrrolidine ring was
determined to be 200S, 500S. Thus, prolinalin A (1) was char-
acterized as 6-C-[(2S,5S)-prolin-5-yl] quercetin.
uration of 2 (Manfre and Pulicani, 1994; Severino et al.,
2003). In association with the 200S, 500R configuration of
2, it is noteworthy that its lower solubility in MeOH and
broadened NMR signals compared to 1 were possibly
due to the presence of intra-molecular hydrogen bonds
between the carboxyl group of the proline moiety and free
hydroxyl groups at C-5 and C-7 of the quercetin moiety.
Such intra-molecular interactions were not present in 1.
From these observations, 2 was determined to be 6-C-
[(2S,5R)-prolin-5-yl] quercetin.
Flavonoids containing nitrogen are very rare natural
products. So far, only eight flavonoidal alkaloids have been
reported: ficine and isoficine from Ficus pantoniana (Johns
et al., 1965), phyllospadine from Phyllosphadix iwatensis
(Takagi et al., 1980), vochsine from Vochysia guaianensis
(Baudouin et al., 1983), lilaline from Lilium candidum
(Masterova et al., 1987), aquiledine and isoaquiledine from
Aquilegia ecalcarata (Chen et al., 2001), and lotthanongine
from Trigonostemon reidioides (Kanchanapoom et al.,
2002). To our knowledge, prolinalin A (1) and B (2) repre-
sent the first examples of a flavonoid conjugated with an
amino acid.
Using an LC mass spectrometer, we attempted to deter-
mine whether the novel flavonoids isolated from the silk-
worm naturally occur in its host plant, M. alba, but
found no evidence of these compounds in the plant. In con-
trast, these flavonoids were found in the cocoon shells of
the silkworm reared on a semi-synthetic diet containing
quercetin (data not shown), indicating that prolinalin A
(1) and B (2) are produced from dietary quercetin within
the insect. A number of lepidopteran insects and some
grasshoppers are known to sequester dietary flavonoids,
and some of these insects have been shown to be able to
glycosylate flavonoid aglycones (Harborne and Grayer,
1994). In larvae of several races of B. mori, in addition to
glycosylation reactions, the dehydration reaction with L-
5-hydroxyproline may be a major pathway of sequestered
flavonoids, however, the physiological roles for the path-
way remain to be studied.
Prolinalin B (2) was observed to be a yellow amorphous
powder as prolinalin A. The high-resolution FT-ICR-MS
measurement of 2 revealed the elementary composition of
protonated ion to be C20H18NO9 ([M + H]+), which is
1
the same as that of 1. The H and 13C NMR (Table 1)
and other spectroscopic data of 2 were very similar to those
of 1, suggesting that the overall structures of 1 and 2 were
the same. Upon acid hydrolysis, 2 also yielded L-d -pyro-
line-5-carboxylic acid, indicative of a 200S configuration.
Therefore, 2 was identified as the diastereoisomer of 1 at
1
the C-500 position of the proline moiety. In the H NMR
spectrum, the H-500 signal of 2 was shifted 0.22 ppm up-
field compared with that of 1, thus suggesting a 500R config-
3. Experimental
OH
3.1. General experimental procedure
OH
3'
H
UV spectra were recorded on a Shimadzu UV-2500 PC
spectrophotometer. High-resolution ESI mass spectra were
obtained by a Bruker Apex II 70e Fourier-transform ion
cyclotron resonance (FT-ICR) mass spectrometer. The
MS/MS experiment was carried out with the FT-ICR mass
spectrometer coupled with an infrared multiphoton dissoci-
1'
O
1
HO
7
H
1''
N
3
5
9.6 Hz
H
OH
10.8 Hz
H
OH
O
HOOC
Hb
1
ation (IRMPD) system. H NMR and 13C NMR spectra
4''
Ha
Ha
were recorded on a Bruker AVANCE 500 spectrometer
(500 MHz for 1H; 125 MHz for 13C) or a Bruker AVANCE
Hb
HMBC correlations
3JH-H couplings
1
800 spectrometer (800 MHz for H; 200 MHz for 13C) in
2.1 Hz
11.1Hz
CD3OD, with TMS as an internal standard. The prepara-
tive and analytical HPLC system consisted of a Shimadzu
3
Fig. 1. Selected HMBC correlations and JH–H couplings of 1.

582
C. Hirayama et al. / Phytochemistry 67 (2006) 579–583
LC-7A pump, a Shimadzu SPD-7AV UV–Visible detector,
and a Shimadzu CTO-10A column oven.
were eluted from the column with a linear gradient of
MeCN in 50 mM triethylamine–phosphate (pH 3.5), from
10% to 40%, for 45 min at a flow rate of 1 ml/min at
25 ꢁC. The FDAA-derivatized L-proline was clearly distin-
guishable from the D-isomer, and its retention time exactly
corresponded to those for the FDAA derivatives of amino
acids derived from 1 and 2.
3.2. Biological material
Daizo, a Chinese race of B. mori is stocked in the
National Institute of Agrobiological Sciences. The larvae
of Daizo were reared on fresh leaves of mulberry M. alba
L. cv. Shin-ichinose planted at the institute. Cocoon shells
produced by the larvae were collected after pupation, then
cut into small pieces.
3.5. Prolinalin A (1)
Yellow amorphous solid. UV (MeOH) kmax nm: 267,
385; 283, 471 (+AlCl3); 279, 440 (+AlCl3 + HCl); 285,
332, 397 (+NaOAc); 275, 402 (+NaOAc + H3BO3). HR-
3.3. Extraction and isolation
1
FT-ICR-MS: 416.0973 (calculated 416.0976). For H, and
Yellow pigments were extracted from the cocoon shell
sample (160 g) by MeOH–H2O (2:1, v/v) at 60 ꢁC for 2 h.
The extract was filtered and concentrated to a small volume
under reduced pressure, after which H2O was added. The
aqueous solution was applied to a solid-phase extraction
cartridge (Oasis HLB, 35 ml, Waters). After washing with
MeOH–H2O (50:50, V/V), the column was eluted with
MeOH–H2O (80:20, V/V). The eluate was concentrated
by evaporation and loaded on a column of Toyoperl
HW-40F (TOSOH). The column was eluted at room
temperature with a linear gradient from 50% to 80%
solvent B (MeOH containing 0.1% formic acid) in solvent
A (0.1% formic acid in H2O). Fractions containing flavo-
noids were concentrated in vacuo and further purified by
reversed-phase HPLC using a Nova-Pak C18 column
(19 · 300 mm, Waters) with a flow rate of 10 ml/min at
40 ꢁC. A 90-min gradient, from 12.5% to 25% solvent B
(solvent A, 0.2% formic acid; solvent B, 0.2% formic acid
in MeCN), was used to afford compounds 1 (tR = 55.2 min,
3.2 mg) and 2 (tR = 51.3 min, 1.5 mg).
13C NMR spectroscopic data, see Table 1.
3.6. Prolinalin B (2)
Yellow amorphous solid. UV (MeOH) kmax nm: 266,
384; 283, 470 (+AlCl3); 279, 444 (+AlCl3 + HCl); 285,
332, 397 (+NaOAc); 275, 401 (+NaOAc + H3BO3). HR-
1
FT-ICR-MS: 416.0962 (calculated 416.0976). For H, and
13C NMR spectroscopic data, see Table 1.
Acknowledgements
We thank Ms. Ikuko Maeda, Dr. Takashi Murata
(Instrumental Analysis Center for Food Chemistry) for
their technical help with the NMR and MS measurements.
We also thank Ms. Mayumi Hazeyama (National Institute
of Agrobiological Sciences) for her technical assistance.
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