Flavonoids from the cocoon of Rondotia menciana

Article abstract of DOI:10.1016/j.phytochem.2013.05.023

Two flavonol glycosides along with four known flavonoids were isolated from the cocoon of the mulberry white caterpillar, Rondotia menciana (Lepidoptera: Bombycidae: Bombycinae), a closely related species of the domesticated silkworm Bombyx mori, both of which feed on leaves of mulberry (Morus alba). The two glycosides were characterized as quercetin 3-O-β-D-galactopyranosyl-(1 → 3)-β-D-galactopyranoside and kaempferol 3-O-β-D- galactopyranosyl-(1 → 3)-β-D-galactopyranoside, based on spectroscopic data and chemical evidence. The flavonol galactosides found in the cocoon were not present in the host plant, nor in the cocoon of the silkworm, B. mori. Notably, flavonol glucosides, which are the main constituents of cocoon flavonoids in B. mori mori, were not found in the R. menciana cocoon. The present result strongly suggests that R. menciana is quite unique in that they predominantly use an UDP-galactosyltransferase for conjugation of dietary flavonoids, whereas UDP-glucosyltransferases are generally used for conjugation of plant phenolics and xenobiotics in other insects.

Full text of DOI:10.1016/j.phytochem.2013.05.023

Phytochemistry 94 (2013) 108–112
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Flavonoids from the cocoon of Rondotia menciana
b
c
d
a
Chikara Hirayama a, , Hiroshi Ono , Yan Meng , Toru Shimada , Takaaki Daimon
⇑
a National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305 8634, Japan
b National Food Research Institute, Tsukuba, Ibaraki 305 8642, Japan
c Anhui Agricultural University, Hefei 230036, China
d The University of Tokyo, Tokyo 113 8657, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 October 2012
Received in revised form 23 February 2013
Available online 3 July 2013
Two flavonol glycosides along with four known flavonoids were isolated from the cocoon of the mulberry
white caterpillar, Rondotia menciana (Lepidoptera: Bombycidae: Bombycinae), a closely related species of
the domesticated silkworm Bombyx mori, both of which feed on leaves of mulberry (Morus alba). The two
glycosides were characterized as quercetin 3-O-b-
D
-galactopyranosyl-(1 ? 3)-b-
D-galactopyranoside and
kaempferol 3-O-b- -galactopyranosyl-(1 ? 3)-b-
D
D-galactopyranoside, based on spectroscopic data and
Keywords:
Morus alba
Moraceae
Cocoon
Flavonol galactosides
Flavonoids
Conjugation
Rondotia menciana
Bombyx mori
chemical evidence. The flavonol galactosides found in the cocoon were not present in the host plant,
nor in the cocoon of the silkworm, B. mori. Notably, flavonol glucosides, which are the main constituents
of cocoon flavonoids in B. mori mori, were not found in the R. menciana cocoon. The present result strongly
suggests that R. menciana is quite unique in that they predominantly use an UDP-galactosyltransferase
for conjugation of dietary flavonoids, whereas UDP-glucosyltransferases are generally used for conjuga-
tion of plant phenolics and xenobiotics in other insects.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
tive studies on ecological aspects of R. menciana and B. mori (e.g.
metabolism of plant secondary metabolites, such as flavonoids
It has been known that cocoon shells of some strains of the
domesticated silkworm, Bombyx mori (L.), contain flavonoids,
which are derived from the leaves of their host plant, the mulberry
tree (Morus alba) (Fujimoto and Hayashiya, 1972; Tamura et al.,
2002; Kurioka and Yamazaki, 2002; Hirayama et al., 2006). Orally
administrated quercetin (5) was preferably glucosylated at the 5-
O-position in the midgut, which is the first step for the biosynthe-
sis of cocoon flavonoids (Hirayama et al., 2008). It was also found
that cocoon flavonoids increase the UV-shielding activity of co-
coons, and protect individuals from harmful effects of sunlight dur-
ing metamorphosis (Daimon et al., 2010).
Uptake and utilization of dietary flavonoids is relatively wide-
spread in the Lepidoptera (Ferreres et al., 2007). Flavonoids seques-
tered by Lepidoptera larvae are subsequently metabolized, stored,
and transferred into the target organs such as the wings (Geuder
et al., 1997). However, as far as is known, there is no report on co-
coon flavonoids of insects other than B. mori. The aim of this study
was to increase the knowledge of cocoon flavonoids of insects. The
mulberry white caterpillar, Rondotia menciana has a close relation-
ship to B. mori, both belonging to the subfamily Bombycinae and
feeding on mulberry leaves. It is considered herein that compara-
and alkaloids) would facilitate an understanding of molecular
mechanisms underlying interactions of insects and plants.
In the present study, cocoon flavonoids of R. menciana were iso-
lated and identified based on spectroscopic methods and chemical
evidence. These results indicated that the main constituents of the
insect cocoon flavonoids were flavonol galactosides. Flavonol
glycosides, which have been found in the cocoon of B. mori and
their host plant, were not detected in the cocoon of R. menciana.
This is the first report suggesting that flavonoids are conjugated
with galactose in animal species, highlighting the complex metab-
olism of flavonoids in insects and its great diversity. This study
further suggests that insect metabolites are a source of potential
bioactive compounds that are not found in plants.
2. Results and discussion
2.1. Structural determination of cocoon shell flavonoids of R. menciana
The HPLC-DAD profile of the aqueous methanolic extract of a
R. menciana cocoon shows six peaks with UV spectra characteristic
of flavonols (Fig. 1A, see also Table S1). These flavonols were
isolated and identified as follows. Acid hydrolysis of peaks a and
⇑
b gave quercetin (5) (Fig. 2) as the aglycone and
as the sugar residue. The UV spectra of both peaks a and b in the
D-galactose (7)
Corresponding author. Tel.: +81 29 838 6152; fax: +81 29 838 6028.
E-mail address: hirayam@nias.affrc.go.jp (C. Hirayama).
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.05.023

C. Hirayama et al. / Phytochemistry 94 (2013) 108–112
109
Fig. 1. Representative HPLC-DAD profiles of aqueous methanolic extracts of R. menciana cocoon (A) and M. alba leaves (B). Detection was performed at 365 nm. a, quercetin 3-
O-galactosyl-galactoside (1); b, quercetin 3-O-galactoside (3); c, kaempferol 3-O-galactosyl-galactoside (2); d, kaempferol 3-O-galactoside (4); e, quercetin (5); f, kaempferol
(6); g, 5-caffeoylquinic acid; h, caffeoylquinic acid; i, quercetin 3-O-rutinoside; j, quercetin 3-O-glucoside; k, quercetin 3-O-(6-malonylglucoside); l, quercetin derivative; m,
kaempferol 3-O-glucoside; n, kaempferol derivative.
Fig. 2. Structures of flavonoids isolated from the cocoon of Rondotia menciana.
presence of shift reagents suggested free hydroxyl groups at C-5, C-
7, C-30 and C-40, and substitution at the 3-O position of quercetin
(5). LC–MS analysis of peaks a and b showed deprotonated ions
[MꢀH]ꢀ with peaks at m/z 625, 463, respectively. Based on these
data, peaks a and b were identified as quercetin 3-O-galactosyl-
galactoside (1) and quercetin-3-O-galactoside (3), respectively.

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C. Hirayama et al. / Phytochemistry 94 (2013) 108–112
After acid hydrolysis of peaks c and d, kaempferol (6) and D-galact-
ose (7) were also obtained. The UV spectra of peaks c and d in the
presence of the shift reagents suggested free hydroxyls at C-5, C-7,
and C-40, and substitution at the 3-O position of kaempferol (6).
LC–MS analysis of peaks c and d showed deprotonated ions
[MꢀH]ꢀ with peaks at m/z 609, 447, respectively. Based on these
data, peaks c and d were identified as kaempferol 3-O-galactosyl-
galactoside (2) and kaempferol 3-O-galactoside (4), respectively.
In addition, peaks e and f were confirmed to be quercetin (5) and
kaempferol (6), by comparing their HPLC-DAD and LC–MS profiles
with those of authentic standards. To determine the detailed
chemical structures of the novel compounds (1 and 2), both com-
pounds were characterized by 1H NMR, 13C NMR, and HR-ESI-MS
analysis. The molecular formula of 1 was determined to be
C
C
27H30O17 by analysis of its HR-ESI-MS spectrum (m/z calcd. for
27H30O17Na ([M+Na]+), 649.13752, found: 649.1375); this corre-
sponds to a digalactosyl derivative of quercetin (5). The 1H NMR
and 13C NMR spectra of 1 (Table 1) were also similar to those of
quercetin 3-O-galactoside (3) (Barberá et al., 1986; Yasukawa and
Takido, 1987; Bennini et al., 1992; Foo et al., 2000). Substitution
at the 3-O position of quercetin (5) was confirmed by the HMBC
correlation between the anomeric proton H-100 (d 5.30) of galactose
and the carbon (d 135.7) of C-3 (Fig. 3). In the 13C NMR spectrum, a
Fig. 3. Key HMBC correlation of 1.
large downfield shift of the galactose C-300 carbon (
D
d11.1ꢁ11.5)
the large coupling constants (7.6–7.9 Hz) at the anomeric position
was observed, compared to the corresponding signal of quercetin
3-O-galactoside (3) (Barberá et al., 1986; Yasukawa and Takido;
1987; Bennini et al., 1992; Foo et al., 2000), suggesting an intergly-
cosidic linkage at this position. This was corroborated by the HMBC
correlation between the anomeric proton H-1000 (d 4.54) of the ter-
minal galactose residue and a carbon (d 84.7) assigned to C00-3 of
the initial galactose residue. It was further confirmed by the HMBC
correlation between the initial galactose H-300 proton (d 3.71) and
the C000-3 carbon of the terminal galactose (d 106.5) (Fig. 3). Further,
the b-configuration of each galactosyl residue was deduced from
in the 1H NMR spectra (Table 1). Therefore, compound 1 was
identified as quercetin 3-O-b-D-galactopyranosyl-(1 ? 3)-b-D-
galactopyranoside.
The structure of 2 was also characterized by the same methods
used in the analysis of 1. Its molecular formula was determined to
be C27H30O16 by HR-ESI-MS spectrum (m/z calcd. for C27H30O16Na
([M+Na]+), 633.14261, found: 633.1431), corresponding to a diga-
lactosyl derivative of kaempferol (6). The 1H NMR and 13C NMR
spectra of 2 were very similar to those of 1, except for signals
assigned to the aglycone moiety. An HMBC experiment indicated
Table 1
1H and 13C NMR spectroscopic data for compounds 1 and 2.
Position
Compound 1
Compound 2
1H d [ppm] multiplicity (J [Hz])
13C d [ppm]
1H d [ppm] multiplicity (J [Hz])
13C d [ppm]
Aglycone
2
3
4
4a
5
6
7
8
158.7
135.7
179.4
105.6
163.0
99.9
159.0
135.5
179.5
105.7
163.1
100.0
165.7
99.7
6.20 d (2.0)
6.40 d (2.0)
6.20 d (2.1)
6.41 d (2.1)
166.2
94.7
8a
10
20
30
40
50
60
158.5
122.9
117.7
145.8
150.0
116.1
122.9
158.5
122.7
132.4
116.1
161.6
116.1
132.4
7.84 d (2.1)
8.09 d (8.9)
6.88 d (8.9)
6.86 d (8.5)
7.58 dd (8.5, 2.1)
6.88 d (8.9)
6.88 d (8.9)
3-O-b-Galp
100
200
300
400
500
600
5.30 d (7.9)
104.7
72.3
84.7
69.6
76.9
62.0
5.29 d (7.9)
104.3
72.2
84.6
69.5
76.8
62.1
3.98 dd (9.6, 7.9)
3.71 dd (9.6, 3.3)
4.16 brd (3.3)
3.53 brdd (6.2, 5.8)
3.67 dd (11.3, 5.8)
3.58 dd (11.4, 6.2)
3.94 dd (9.6, 7.9)
3.70 dd (9.6, 3.3)
4.11 dd (3.3, 0.9)
3.50 ddd (6.4, 5.8, 0.9)
3.64 dd (11.3, 5.8)
3.54 dd (11.4, 6.4)
300-O-b-Galp
1000
2000
3000
4000
5000
6000
4.54 d (7.6)
106.5
73.0
74.6
70.2
76.8
62.6
4.52 d (7.6)
106.4
73.0
74.6
70.2
76.8
62.6
3.64 dd (9.7, 7.6)
3.51 dd (9.7, 3.3)
3.83 brd (3.3)
3.55 brdd (7.1, 5.0)
3.74 dd (11.4, 7.1)
3.70 dd (11.4, 5.0)
3.63 dd (9.7, 7.6)
3.50 dd (9.7, 3.3)
3.82 dd (3.3, 0.8)
3.54 ddd (7.1, 5.2, 0.8)
3.74 dd (11.4, 7.1)
3.69 dd (11.4, 5.2)

C. Hirayama et al. / Phytochemistry 94 (2013) 108–112
111
that the residual galactose was attached at the 3-O position of the
kaempferol (6), and that the other galactose residue was attached
to the 300-O position of the initial galactose. These data identified
conjugated with galactose in animal species. The present results
contribute the understanding the complex metabolism of flavo-
noids in insects.
compound 2 as kaempferol 3-O-b-D-galactopyranosyl-(1 ? 3)-b-
D
-galactopyranoside.
4. Experimental
4.1. Chemicals and reagents
2.2. Host plant flavonoids uptake and metabolism by R. menciana
Quercetin 3-O-glucoside, kaempferol 3-O-glucoside, rutin, quer-
cetin 3-O-galactoside (3), quercetin (5), and kaempferol (6) were
from Extrasynthese (Genay, France). Qurcetin 3-O-(6-malonylg-
lucoside) was a gift from Dr. Takuya Katsube (Shimane institute
for industrial technology, Shimane, Japan). A galactose assay kit
was purchased from Biovision (Milpitas, CA), whereas a, glucose
assay kit was from Wako pure chemical (Osaka, Japan). All other
chemicals and solvents used were of either analytical or HPLC
grade. Water (H2O) was treated with a Milli-Q water purification
system (Millipore, Bedford, MA).
The flavonoids of mulberry (M. alba) leaves have been isolated
and characterized by many researchers. These study described that
mulberry leaves contained quercetin 3-O-glucoside, quercetin
3-O-rutinoside, quercetin 3-O-(6-malonylglucoside), quercetin
3-O-(6-acetylglucoside), kaempferol 3-O-glucoside, kaempferol
3-O-(6-acetylglucoside), kaempferol 3-O-(6-malonylglucoside) (Naito,
1968, 1979; Onogi et al., 1994; Doi et al., 2001; Katsube et al., 2006,
2009). The present study confirmed that the main constituent of
the mulberry leaves was quercetin 3-O-(6-malonylglucoside),
followed by quercetin 3-O-rutinoside and quercetin 3-O-glucoside
(Fig. 2B). Comparison of the flavonoid profile of R. menciana with
that of mulberry established that there were no compounds in
common for the insect and its host plant (Fig. 1, see also
Table S1). Interestingly, even though no flavonol galactosides have
been found from the food source plant, flavonol galactosides were
the main constitutes of the cocoon extracts (Fig. 1A, see also
Table S2). This can be ascribed to metabolism of the flavonols in
the insect. It is thus considered that flavonol glycosides derived
from the mulberry are hydrolyzed to aglycones (quercetin (5)
and kaempferol (6)) during uptake by the midgut tissue, then con-
jugated to galactose for further transport to the silk gland, where
the raw materials for cocoon shells are produced. Schittko et al.
(1999) showed that quercetin 3-O-galactoside (3) was the main
component of flavonoids accumulated in the common blue butter-
fly, Polyommatus icarus. However, P. icarus larvae probably uptake
and store quercetin 3-O-galactoside (3) without modification since
inflorescences of Trifolium repens, the food plant source of P. icarus,
is also rich in quercetin 3-O-galactoside (3) (Schittko et al., 1999).
Thus, so far, there have been no reports on galactosyl-conjuga-
tion reaction of plant phenolics in animal tissues. In general, in-
sects have been known to use UDP-glucosyltransferase to modify
plant phenolics and xenobiotics (Ahmad and Hopkins, 1993; Ahn
et al., 2011; Rausell et al., 1997; Real et al., 1991; Sasai
et al.,2009). It was found, however, that B. mori also uses UDP-glu-
cosyltransferase in the metabolism of dietary flavonoids (Hirayama
et al., 2008; Daimon et al., 2010). Although it is necessary to show
the actual enzyme activity in the near future, the present study
suggests that R. menciana uses UDP-galactosyltransferase instead
of UDP-glucosyltransferase. It is very interesting to now consider
why R. menciana uses an UDP-galactosyltransferase, even though
R. menciana and B. mori are closely related and reared on the same
food plant. Further investigation of the biological functions of fla-
vonol galactosides found in R. menciana may help to understand
its evolution, as well as the ecological significance of the galactosyl
conjugation reaction of flavonoids in insects, i.e. thereby poten-
tially constituting a source of potential bioactive compounds not
found so far. In addition, molecular and biochemical analysis of
the UDP-galactosyltransferase of R. menciana could be useful for
understanding the molecular basis of substrate specificity of
UDP-glycosyltransferase (UGT) family enzymes in animals.
4.2. Biological materials
R. menciana were maintained in the Anhui Agricultural Univer-
sity, the National Institute of Agrobiological Sciences, and the Uni-
versity of Tokyo. The larvae of R. menciana were reared on fresh
leaves of mulberry (M. alba L). Cocoon shells produced by the lar-
vae were harvested after adult emergence, then cut into small
pieces and stored at ꢀ80 °C until needed. Collected mulberry
leaves were frozen, and then lyophilized. Powdered mulberry sam-
ples were kept at ꢀ80 °C until analysis.
4.3. LC–MS analysis
LC–ESI-MS analysis was performed using a HP 1100 series HPLC
(Agilent Technologies, Santa Clara, CA) equipped with an HP 1100
MSD mass spectrometer. Flavonoids were extracted from either co-
coon shells or lyophilized mulberry powder by MeOH–H2O (7:3, V/
V) at 60 °C for 2 h. After centrifugation at 20,000g for 10 min, an
aliquot (10 ll) of the supernatants was injected into the LC–MS
system and separated by a Nova-Pak C18 reversed phase column,
150 mm ꢂ 2.0 mm i.d. (Waters, Milford, MA) at a flow rate of
0.3 ml/min. Column temperature was maintained at 40 °C. The mo-
bile phase consisted of solvents A (0.2% aq. HCO2H formic acid) and
B (0.2% HCO2H in CH3CN). Flavonoids were separated with a linear
gradient from 7% B to 40% B over 40 min and then to 100% B for
5 min. UV detection was carried out using a HP 1100 photodiode
array detector (DAD) to facilitate peak assignment. The eluent
was ionized by negative electrospray ionization. The electrospray
mass spectrometer conditions were as follows: negative ion mode;
fragmentor voltage, 70 V; capillary voltage, 3500 V; drying gas (N2)
flow, 10.0 L/min; nebulizer pressure, 25 psig; drying gas tempera-
ture, 350 °C. Some flavonoids were identified based on their co-
elution with commercially available standards and their MS
profile.
4.4. Isolation of flavonoids from the cocoon shell of R. menciana
Flavonoids were extracted from cocoon shells (10 g) using
MeOH–H2O (7:3, V/V) at 60 °C for 2 h. Crude extracts were filtered
and concentrated by evaporation and diluted with H2O. Each di-
luted solution was applied to a solid phase extraction cartridge
(Oasis HLB, 35 ml, Waters, Milford, MA). After washing with H2O,
the column was eluted with MeOH. Eluted flavonoids were concen-
trated by evaporation and loaded to a preparative reversed-phase
3. Conclusion
Cocoon flavonoids of R. menciana were isolated and identified
based on spectroscopic methods and chemical evidence, indicating
that the main constituents of the cocoon flavonoids are flavonol
galactosides. This is the first report suggesting that flavonoids are
column (Sunfire C18, 19 ꢂ 150 mm, Waters) connected to
a
a
Shimadzu HPLC system equipped with an LC-7A pump,

112
C. Hirayama et al. / Phytochemistry 94 (2013) 108–112
CTO-10A column oven, and an SPD-7AV UV–Vis detector monitor-
ing at 365 nm. Flavonoids were eluted by a linear gradient of 14–
25% CH3CN in 0.2% HCO2H over 90 min, at a flow rate of 10 ml/
min at 40 °C.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2013.
05.023.
4.5. Sugar and aglycone analysis
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Yellow powder; UV kmax nm; 267, 351 (MeOH); 275, 326, 403
(+NaOMe); 275, 376 (+NaOAc); 267, 353 (+NaOAc + H3BO3); 274,
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We thank Dr. Ikuko Maeda, Ms. Tomoko Sato (Instrumental
Analysis Center for Food Chemistry, NARO) for their technical help
with NMR and MS measurements. We also thank Ms. Mayumi
Hazeyama (National Institute of Agrobiological Sciences) for tech-
nical assistance. This study was partly supported by grants-aid for
scientific research (No. 23688008) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
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