A new 2'0-oxygenated flavone glycoside from litsea glutinosa (Lour.) C. B. Rob.

Article abstract of DOI:10.1271/bbb.90701

A new 20-oxygenated flavone glycoside, named glutin, was isolated from Litsea glutinosa (Lour.) C. B. Rob. along with four known compounds. The structure was identified as 20,5,7-trihydroxy-6-methoxyflavone 20-O-Β-D-glucopyranoside (1) on the basis of extensive spectroscopic analysis.

Full text of DOI:10.1271/bbb.90701

Biosci. Biotechnol. Biochem., 74 (3), 652–654, 2010
Note
A New 20-Oxygenated Flavone Glycoside
from Litsea glutinosa (Lour.) C. B. Rob.
y
Yun-Song WANG, Rong HUANG, Hao LU, Feng-Ya LI, and Jing-Hua YANG
Key Laboratory of Medicinal Chemistry for Natural Resources, Ministry of Education,
School of Chemical Science and Technology, Yunnan University, Kunming 650091, P. R. China
Received September 28, 2009; Accepted December 11, 2009; Online Publication, March 7, 2010
[doi:10.1271/bbb.90701]
A new 20-oxygenated flavone glycoside, named glutin,
was isolated from Litsea glutinosa (Lour.) C. B. Rob.
along with four known compounds. The structure was
identified as 20,5,7-trihydroxy-6-methoxyflavone 20-O-ꢀ-
D-glucopyranoside (1) on the basis of extensive spectro-
scopic analysis.
ether-EtOAc, and crystals from Fraction III were puri-
fied by recrystallization (CHCl3–MeOH 1:1) to give
compounds 5 (38 mg). Fraction IV was resubmitted to
Sephadex LH-20 (using MeOH as eluent) and RP C-18
(MeOH–H2O 7:3, 0.7 liter; 6:4, 0.5 liter) to yield
compounds 1 (8 mg). Fraction V was rechromato-
graphed on a silica gel column, and eluted with CHCl3
containing increasing amounts of MeOH, and then to
Sephadex LH-20 column chromatography (using ace-
tone as eluent) to give compounds 4 (12 mg).
Key words: flavonoids; Litsea glutinosa (Lour.) C. B.
Rob.; glutin
The genus Litsea (Lauraceae) comprises nearly 200
species, which are distributed widely throughout tropical
and subtropical Asia, North America and subtropical
South America. Previous phytochemical studies re-
vealed the presence of alkaloids,1–3) butanolides,4,5) and
sesquiterpenoid in the genus Litsea.6–9) Few records are
found in the literature with respect to the isolation of
flavonoids from Litsea species.10)
Compound 1 was isolated as a yellow amorphous
powder. A positive Shinoda and Fiegel’s test indicated
that compound 1 was a flavonoid glycoside.14) Its
formula, C22H22O11, was derived from positive HR-
ESI-MS measurements of the molecular-related ion at
m=z 485.1053 (calcd. for 485.1059) ½M þ Naꢀþ, and
from 13C and DEPT NMR data. The IR spectrum (KBr)
of 1 showed a hydroxyl absorption band at 3,435 cmꢁ1
and a carbonyl absorption band at 1,658 cmꢁ1. The UV
spectrum showed ꢀmax at 208, 267, and 326 nm (MeOH).
A bathochromic shift (ꢀmax 326, 267, 215 nm) of 7 nm of
band II with NaOAc indicated the presence of a free
7-OH group. Several strong, broad bands in the range of
1,650–1,051 cmꢁ1 in the IR spectrum indicated a flavone
skeleton. A prominent fragment at m=z 301 ½M þ H ꢁ
Litsea glutinosa (Lour.) C. B. Rob., locally known as
‘‘Chan Gao Shu’’ in China, is an evergreen medium-
sized tree. Its bark and leaves are used as a demulcent
and mild astringent for diarrhea and dysentery, and the
roots are used to poultice sprains and bruises.11) To date,
there have been few phytochemical studies on the
species Litsea glutinosa (Lour.) C. B. Rob.12) Our
previous investigation resulted in the isolation of two
new aporphine alkaloids from this species.13) As part
of continuous research on plants of the Litsea genus,
the chemical constituents of the leaves and twigs of
L. glutinosa (Lour.) C. B. Rob. were reinvestigated.
Powdered leaves and twigs of Litsea glutinosa (Lour.)
C. B. Rob. (12.0 kg) were repeatedly extracted with
EtOH at room temperature. The extract was then
concentrated under reduced pressure to give a brown
syrup, which was partitioned in H2O and extracted with
solvents into a petroleum ether-fraction (80 g), an
EtOAc-fraction (54 g), and an n-BuOH-fraction (108 g)
fraction. The petroleum-ether fraction was subjected to
silica gel column chromatography eluted with petroleum
ether-EtOAc (10:1–1:1), by which five fractions (I–V)
were obtained. Fraction II was resubmitted to silica gel
column chromatography to yield compounds 2 (20 mg)
and 3 (55 mg). The EtOAc-fraction was subjected to
silica gel column chromatography eluted with CHCl3–
MeOH (99:1–1:1) to afford eight fractions (I–VIII).
Fraction III was resubmitted to silica gel column
chromatography by gradient elution using petroleum
162ꢀþ and the H and 13C NMR spectra indicated the
1
presence of a glucopyranosyl unit. Glycoside 1 (1 mg)
was hydrolyzed with 2 N HCl at 100 ꢂC for 40 min. The
reaction mixture was diluted with water and extracted
with EtOAc. The sugar in the aqueous layer was
identified as D-glucose by co-PC using solvent system
n-BuOH–HOAc–H2O (4:1:5).
The 1H NMR spectrum exhibited the presence of
a sharp single hydroxyl signal at ꢁH 13.0 and another
hydroxyl signal, ꢁH 10.75, that indicated that the A-ring
had a 5,7-dihydroxy substituted pattern (Table 1). The
aromatic region of the 1H NMR spectrum of 1 displayed
a sharp one-proton singlet signal at ꢁH 7.01, correlated
with C-3 (109.8) in its HMQC spectrum, which is
characteristic the C-3 proton of 20-oxygenated fla-
vone.15) Likewise, a second singlet resonance in the
aromatic region at ꢁH 6.58 (1H, ꢁC 94.3 by HMQC) was
assigned to H-8 on the basis of long-range connectivities
to ꢁC 157.5 (C-7), 152.8 (C-9), 131.4 (C-6), and 104.2
(C-10). A methoxyl signal at ꢁH 3.76 was placed at
3
C-6 based on J correlation of these protons with C-6
at ꢁC 131.4 in the HMBC spectrum. The presence of
y
To whom correspondence should be addressed. Tel: +86-871-6598387; Fax: +86-871-5035538; E-mail: yangjh@ynu.edu.cn; yangjhkm@-
hotmail.com

New 20-Oxygenated Flavone Glycoside from Litsea glutinosa
653
Table 1. 13C- (125 MHz) and 1H- (500 MHz) NMR Data for 1 (in DMSO-d6)
Position
ꢁC
ꢁH (integral, mult, J in Hz)
Position
ꢁC
ꢁH (integral, mult, J in Hz)
2
3
160.9
109.8
182.3
152.7
131.4
157.5
94.3
/
7.01(1H, s)
40
50
132.9
122.0
129.1
100.2
73.4
7.56 (1H, ddd, 1.6, 7.6, 7.8 Hz)
7.23 (1H, ddd, 1.6, 7.6, 7.6)
7.89 (1H, dd, 1.6, 7.6 )
5.12 (1H, d, 7.1)
4
/
13.0 (1H, s, –OH)
60
5
20-Glu:100
200
6
/
10.75 (1H, s, –OH)
3.36 (1H, m)
3.37 (1H, m)
7
300
76.8
69.6
8
6.58 (1H, s)
/
400
3.29 (1H, m)
3.40 (1H, m)
9
152.8
104.2
120.3
155.4
115.5
500
77.2
10
10
20
30
/
600a
600b
6-OMe
3.47 (1H, dd, 14.6, 4.8)
3.70 (1H, dd, 14.6, 5.5)
3.76 (3H, s)
60.6
60.0
/
/
7.36 (1H, dd, 1.6, 7.8)
5'
Selected HMBC and NOESY of 1
4'
3'
6'
8
HO
O
2
9
HO
O
7
2'
3
O
10
6
MeO
4
5
OH
MeO
O
OH
OH
O
OH
O
OH
OH
O
O
1
HMBC
H
H
C
H
HO
OH
OH
NOESY
HO
Fig. 1. The Structure, Key HMBC Correlations, and Selected NOESY of 1.
7-hydroxy and 6-methoxyl groups was also confirmed
by comparison of C-6 (ꢁC 131.4) and C-8 (ꢁC 94.3)
13C NMR signals of 1 with those (ꢁC 131.5, 94.5) of
5,7-dihydroxy-6-methoxyflavone.16)
Our preliminary pharmacological study of this plant
showed the EtOAc extract to have cytotoxic activity
against human Hela (human carcinoma of the cervix)
cell lines at a concentration of 10 mg/ml. But compound
1 did not show potential in vitro cytotoxicity screening
against human Hela cell lines. Besides, 1 was screened
for anti-oxidant activities using DPPH radicals by the
described method.21) In brief, 5 ml of test sample (1 mM
in DMSO) was seeded to 95 ml of 300 mM ethanolic
DPPH and incubated at 37 ꢂC. After 30 min, the
absorbance of the mixture was measured at 517 nm in
a microplate reader. Percent radical scavenging activity
was determined by comparison with a DMSO-contain-
ing control. IC50 values represent concentrations of
compounds scavenging 50% of DPPH radicals. BHA
(3-t-butyl-4-hydroxyanisole) was used as a positive
control. The results displayed that 1 scavenged DPPH
radicals (53.5%), while standard anti-oxidant BHA as a
positive control was active at a level of 92.1% at 1 mM.
The four spin-spin coupled multiplets at ꢁH 7.89
(1H, dd, J ¼ 7:6, 1.6 Hz), 7.56 (1H, ddd, J ¼ 1:6, 7.6,
7.8 Hz), 7.36 (1H, dd, J ¼ 1:6, 7.8 Hz), and 7.23 (1H,
ddd, J ¼ 7:6, 7.6, 1.6 Hz) comprised the remaining
aromatic resonances and are characteristic ABCD signal
pattern of a 20-oxygenated B-ring,15) and were assigned
to the C-60, C-40, C-30, and C-50 proton, respectively.
The HMBC spectrum was utilized to identify the
position of the sugar. A long-range correlation between
the anomeric proton signal (ꢁH 5.12) of glucose and C-20
(ꢁC 155.4) confirmed the attachment of the sugar at C-20.
Further useful correlations from H-30 to 120.3s (C-10),
155.4s (C-20), H-40 to 129.1d (C-60), 155.4s (C-20), and
H-60 to 132.9d (C-40), 155.4s (C-20), and 160.9s (C-2)
confirmed this. The NOESY spectrum of 1 gave a cross
peak between the H-30 signal and the anomeric proton
signal (ꢁH 5.12) of glucose, indicating further that the
glucose moiety in 1 is linked to the C-20 hydroxy group.
The coupling constant (J ¼ 7:1 Hz) of the anomeric
proton signal indicated the ꢂ-configuration of the
glucopyranoside moiety. Thus, the new flavone glyco-
side was identified as 20,5,7-trihydroxy-6-methoxyfla-
vone 20-O-ꢂ-D-glucopyranoside, and was named glutin
(Fig. 1).
Acknowledgments
This work was supported by the National Nature
Science Foundation of China (no. 20862018), the
Science Foundation of Yunnan University (Grant no.
2009B10Q and 2005Z001A), and the Science Founda-
tion of Yunnan Province (Grants nos. 2006B0003Q,
2007B0006Z, and 2007PY01-23).
The structures of known compounds 2–5 were
identified as sitosterol (2),17) stigmasterol (3),18) epica-
techin (4),19) and sitosterol ꢂ-D-glucopyranoside (5)20)
by a combination of spectroscopic analysis and compar-
ison with the literature data. Compound 1 was charac-
terized as a 20-oxygenated flavone glycoside; this is the
first time that a C-20 oxygenated flavonoid has been
isolated from the genus Litsea.
References
1) Hart NK, Johns SR, Lamberton JA, Loder JW, Moorhouse A,
Sioumis AA, and Smith TK, Aust. J. Chem., 22, 2259–2262
(1969).
2) Bhakuni DS and Gupta S, Planta Med., 48, 52–54 (1983).
3) Holloway DW and Scheinmann F, Phytochemistry, 12, 1503–
1505 (1973).

654
Y.-S. WANG et al.
4) Cheng HI, Lin WY, Duh CY, Lee KH, Tsai IL, and Chen IS,
J. Nat. Prod., 64, 1502–1505 (2001).
12) Herath HMTB, Kumar NS, and Wimalasiri KMS, Carbohydr.
Res., 198, 343–351 (1990).
5) Chen IS, Lai Yaun IL, Duh CY, and Tsai IL, Phytochemistry,
49, 745–750 (1998).
13) Yang JH, Li L, Wang YS, Huang R, Zhao JF, and Luo SD, Helv.
Chim. Acta, 88, 2523–2526 (2005).
6) Hoang VD, Tan GT, Zhang HJ, Tamez PA, Hung NV, Cuong
NM, Soejarto DD, Fong HHS, and Pezzuto JM, Phytochemistry,
59, 325–329 (2002).
14) Grayer RJ, ‘‘Methods in Plant Biochemistry: Flavonoids’’ Vol. 1,
eds. Dey PM and Harbone JB, Academic Press, London, p. 292
(1989).
7) Zhang HJ, Tan GT, Hoang VD, Hung NV, Cuong NM, Soejarto
DD, Pezzuto JM, and Fong HHS, Tetrahedron Lett., 42, 8587–
8591 (2001).
15) Tanaka T, Iinuma M, and Mizumo M, Chem. Pharm. Bull., 34,
1667–1670 (1986).
16) Iwashina T, Kamenosono K, and Ueno T, Phytochemistry, 51,
1109–1111 (1999).
8) Zhang HJ, Tan GT, Hoang VD, Hung NV, Cuong NM, Soejarto
DD, Pezzuto JM, and Fong HHS, Tetrahedron, 59, 141–148
(2003).
17) Dan S and Dan SS, Fitoterapia, 53, 51–52 (1982).
18) Zhang XQ, Qi J, Ye WC, and Zhao SX, J. China Pharm. Univ.
(in Chinese), 35, 404–405 (2004).
9) Zhang HJ, Tan GT, Santarsiero BD, Mesecar AD, Hung NV,
Cuong NM, Soejarto DD, Pezzuto JM, and Fong HHS, J. Nat.
Prod., 66, 609–615 (2003).
19) Lee SY, Min BS, Kim JH, Lee J, Kim TJ, Kim CS, Kim YH,
and Lee HK, Phytother. Res., 19, 273–276 (2005).
20) Flamini G, Antognoli E, and Morelli I, Phytochemistry, 57,
559–564 (2001).
10) Wang JQ, Li J, Zou YH, Cheng WM, Lu C, Zhang L, Ge JF,
Huang C, Jin Y, Lu XW, Hu CM, and Liu LP, J. Ethno-
pharmacol., 121, 54–60 (2009).
21) Na MK, An RB, Jin WY, Min BS, Yoo JK, Kim YH, and Bae
K, Nat. Prod. Sci., 9, 226–231 (2003).
11) Li XW, ‘‘Flora Reipublicae Popularis Sinicae’’ (in Chinese),
tomus 31, Science Press, Beijing, p. 285 (1982).