A new flavonoid glycoside and four other chemical constituents from viscum coloratum and their antioxidant activity

Article abstract of DOI:10.3987/COM-14-12934

A new flavonoid glycoside, identified as rhamnazin-3-β-D-(6″- β- hydroxy-β-methylglutaryl)-β-D-glucoside-4′-β-D- glucoside (1), along with four known compounds, shikimic acid (2), quebrachitol (3), abscisic acid (4), 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane (5), were isolated from the branches and leaves of Viscum coloratum. Their structures were elucidated on the basis of MS, 1D NMR, and 2D NMR techniques. All compounds were isolated in the family Loranthaceae for the first time. This paper describes the isolation, structural elucidation as well as in vitro antioxidant activity of these isolates.

Full text of DOI:10.3987/COM-14-12934

HETEROCYCLES, Vol. 89, No. 6, 2014
1455
HETEROCYCLES, Vol. 89, No. 6, 2014, pp. 1455 - 1462. © 2014 The Japan Institute of Heterocyclic Chemistry
Received, 8th January, 2014, Accepted, 11th April, 2014, Published online, 23rd April, 2014
DOI: 10.3987/COM-14-12934
A NEW FLAVONOID GLYCOSIDE AND FOUR OTHER CHEMICAL
CONSTITUENTS FROM VISCUM COLORATUM AND THEIR
ANTIOXIDANT ACTIVITY
Ronghua Fan,a Yuying Ma,a Hongxia Yuan,b Yongzhi Zhang,a Binbin Wei,a
Yunli Zhao,a,* and Zhiguo Yua,*
a: School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016,
China; E-mail address: zhiguo-yu@163.com; Yunli76@163.com
b: Shanxi University of Traditional Chinese Medicine, Taiyuan, 030024, China
Abstract – A new flavonoid glycoside, identified as rhamnazin-3-Ο-β-D-(6′′-β-
hydroxy-β-methylglutaryl)-β-D-glucoside-4′-Ο-β-D-glucoside (1), along with four
known compounds, shikimic acid (2), quebrachitol (3), abscisic acid (4),
3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane (5), were isolated from the
branches and leaves of Viscum coloratum. Their structures were elucidated on the
basis of MS, 1D NMR, and 2D NMR techniques. All compounds were isolated in
the family Loranthaceae for the first time. This paper describes the isolation,
structural elucidation as well as in vitro antioxidant activity of these isolates.
The Viscum coloratum, also referred to as “mistletoe”, is an evergreen, semi-parasitic plant which grows
on the branches or stems of deciduous trees. The branches and leaves of mistletoe are used in the
treatment of hypertension, atherosclerosis, rheumatism and neuralgia.1-3 The activity of mistletoe has been
ascribed to the presence of antioxidative constituents.4,5 Therefore, the bioactive constituents of V.
coloratum are of our interest. The ethanol extract of mistletoe showed potent antioxidative properties in
our screening program searching for antioxidative natural substances, and further bioassay-guided
analysis led to the isolation of a new flavonoid glycoside and four known compounds from the active
antioxidative extract. All the five compounds were characterized in the family Loranthaceae for the first
time. In this paper, we report the isolation, structure elucidation and antioxidative activities of these

1456
HETEROCYCLES, Vol. 89, No. 6, 2014
compounds. Their antioxidant activity was tested in vitro by the DPPH scavenging method.
The air-dried stems and leaves of mistletoe, collected in Liaoning province of China, were extracted
exhaustively with 95% EtOH under reflux. Repeated chromatographic separation of the combined
extracts led to the purification and subsequent characterization of a new flavonoid glycoside (1), along
with known compounds shikimic acid (2), quebrachitol (3), abscisic acid (4), and
3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane (5).
OMe
3'
2'
8
MeO
O
O
1'
2
4'
O
7
6
3
5'
6'''
6'
4
O
5
OH
HO
4'''
5'''
O
2'''
O
OH
1'''
O
HO
OH
C
5''''
3'''
C
6''
5''
OH
HO
3''''
1''''
O
4''''
2''''
O
2''
OH
4''
1''
OH
3''
OH
Figure 1. The structure of compound 1
Compound 1 was obtained as yellow powder. The structure was shown in Figure 1. Its HR-ESI-MS
spectrum displayed a [M + H]+ ion peak at m/z 799.2295, indicating a molecular formula of C35H42O21
(calcd. for 799.2297) (fifteen degrees of unsaturation). Its IR spectrum indicated the presence of hydroxy
(3420 cm-1), methyl (2924 cm-1), carbonyl (1729 cm-1) and phenyl (1654 cm-1, 1600 cm-1, 1071 cm-1)
groups. The 1H and 13C NMR signals of compound 1 were assigned by different 2D NMR analysis. The 1H
NMR spectrum of compound 1 showed the presence of three protons at δ 1.14 (3H, s) predicting 3′′′′-Me,
four protons at δ 2.42 (2H, m) and 2.39 (2H, m) corresponding to H2-2′′′′ and H2-4′′′′, two methyl signals
at δ 3.94 (3H, s) and δ 3.88 (3H, s) corresponding to 3′-OMe and 7-OMe respectively. Anomeric protons
resonating as a doublet at δ 5.33 (1H, d, J = 5.4 Hz) (δ 5.50 (1H, d, J = 7.2 Hz in DMSO-d6)) and another
at δ 5.05 (1H, d, J = 7.2 Hz) indicated the presence of β-glucose, respectively. The presence of a flavonol
A-ring was confirmed by two mutually coupled protons at δ 6.33 (1H, s, H-6) and δ 6.61 (1H, s, H-8).
The presence of flavonol B-ring was confirmed by three mutually coupled protons at δ 7.92 (1H, s, H-2′),
7.25 (1H, d, J = 13.2 Hz, H-5′) and 7.69 (1H, d, J = 8.4 Hz, H-6′). There were 35 carbon signals in the 13C
NMR spectrum of compound 1. The signals at δ 172.3 (C-1′′′′), 46.4 (C-2′′′′), 70.6 (C-3′′′′), 46.1 (C-4′′′′),
175.8 (C-5′′′′) and 27.6 (3′′′′-C) corresponded to the carbon signals of 3-hydroxy-3-methylglutarate. The

HETEROCYCLES, Vol. 89, No. 6, 2014
1457
signals at δ 55.6 and δ 57.0 corresponded to the -OMe of C-7 and C-3′, respectively. The signals at δ
103.7 and δ 102.1 were C-1′′and C-1′′′ signals of glucose. The carbon signals left were corresponded to
the flavone skeleton and glucose. In the HMBC spectrum (Figure 2), the proton at δ 1.14 (3H, s, 3′′′′-Me)
showed 3J-correlation with C-2′′′′ and C-4′′′′; the 2J-correlation with C-3′′′′ indicated that the methyl
group was situated at C-3′′′′. The proton at δ 4.20 (1H, d, 6′′-H) and δ 4.09 (1H, dd, 6′′-H) showed
3J-correlation with C-1′′′′. The proton at δ 5.33 (1H, d, 1′′-H) and δ 5.05 (1H, d, 1′′′-H) showed
correlation with C-3 and C-4′ respectively. The TOCOSY correlation of H-1′′/H-4′′and the HMBC
correlation from H-6′′ (δH 4.09) to C-4′′ indicated that the 3-hydroxy-3-methylglutarate group was
situated at glucose 1. The 1H and 13C NMR spectroscopic data were listed in Table 1. The complete acid
hydrolysis of compound 1 yielded arious products, and in the hydrolysate separated from the aglycone
parts, the sugar was identified as glucose by the thin layer chromatography (TLC) comparison with the
standard sample. The D-glucose could be confirmed by the optical rotation of [α]2D0 +12 (c 0.02, H2O).6
Consequently,
the
structure
of
compound
1
was
assigned
as
rhamnazin-3-Ο-β-D-(6′′-β-hydroxy-β-methylglutaryl)-β-D-glucoside-4′-Ο-β-D-glucoside.
1
13
Table 1. The H NMR (600 MHz) and C NMR (150 MHz) spectral data and HMBC correlations of
compound 1 (in methanol-d4, δ ppm, J in Hz)
Position
δ H
δ C
HMBC(H→C)
2
158.5
135.9
179.5
162.8
99.3
3
4
5
6
6.33
6.61
C-5, 7, 8, 10
C-6, 7, 9, 10
7
167.5
93.3
8
9
158.4
106.7
123.2
114.9
10
1′
2′
7.92
C-2, 1′, 3′, 4′, 5′

1458
HETEROCYCLES, Vol. 89, No. 6, 2014
3′
150.0
150.4
4′
5′
7.25 (d, 13.2)
116.6
125.8
57.0
C-3′, 4′, 6′
C-2, 2′, 4′, 5′
C-7
6′
7.69 (d, 8.4)
3.88
7-OCH3
3′-OCH3
Glc 1
1′′
3.94
56.6
C-3′
5.33 (d, 5.4)
3.45 (m)
103.7
75.8
77.8
71.6
75.8
64.4
C-3
2′′
C-3′′
3′′
3.45 (m)
C-1′′, 2′′, 5′′
C-2′′, 3′′, 5′′
C-3′′
4′′
3.28 (m)
5′′
3.45 (m)
6′′
4.20 (d, 12)
4.09 (dd, 10.8, 6)
C-1′′′′, 4′′, 5′′
Glc 2
1′′′
5.05 (d, 7.2)
3.55 (m)
3.45 (m)
3.43 (m)
3.50 (m)
3.91 (m)
3.72 (m)
102.1
74.8
77.8
71.3
78.3
62.5
C-4′
2′′′
C-1′′′, 3′′′
3′′′
4′′′
C-2′′′
5′′′
C-2′′′, 3′′′, 4′′′
6′′′
C-4′′′, 5′′′
3-Hydroxy-3-methylglutarate
1′′′′
172.3
46.4
70.6
46.1
175.8
27.6
2′′′′
2.42 (m)
2.39 (m)
1.14
C-1′′′′, 3′′′′, 4′′′′, 5′′′′
C- 2′′′′, 3′′′′, 5′′′′
C-3′′′′, 4′′′′
3′′′′
4′′′′
5′′′′
3′′′′-CH3

HETEROCYCLES, Vol. 89, No. 6, 2014
1459
OMe
O
MeO
O
O
HO
OH
O
O
O
C
OH
OH
O
C
HO
OH
HO
O
O
OH
OH
OH
Figure 2. Key HMBC correlations of compound 1
The four known compounds were determined as shikimic acid,7 quebrachitol,8 abscisic acid,9
3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane,10,11 by comparison of their spectral data with literature
values.
The potential antioxidant activity of the compounds was assessed in vitro and expressed as EC50 values
by the DPPH scavenging method. The results of DPPH radical scavenging activities for all isolates are
summarized in Table 2. All tested compounds showed moderate antioxidant activity.
Table 2. The results of the antioxidant activities of the five compounds and Vitamin C
Compounds
VC
1
2
3
4
5
EC50 values (μ mol/mL) 0.068
0.440
0.612
1.550
0.365
0.417
EXPERIMENTAL
General
UV absorption spectra were measured on Shimadzu UV-1700 Spectrophotometer and Perkin Elmer
Lambda 35 UV/VIS Spectrometer. IR absorption spectra were recorded on a Bruker IFS 55 Infrared
spectrophotometer. NMR spectra were measured with 600 MHz Bruker FTNMR Ultra ShieldTM
spectrometers in methanol-d4. Chemical shifts (δ ppm) were given relative to TMS as internal standard.
HR-ESI-MS was performed on Waters Xevo G2 QTOF MS. Optical rotations were measured with a
Perkin-Elmer 241 MC polarimeter. Column chromatography was performed using silica gel (Qingdao

1460
HETEROCYCLES, Vol. 89, No. 6, 2014
Haiyang Chemical Group Co., Ltd., Qingdao, Shandong, China), polyamide (Luqiao Sijia Biochemical
plastic factory, Taizhou, Zhejiang, China), Sephadex LH-20 (GE Healthcare, Piscataway, NJ, USA) and
ODS (Phenomenex Inc., Torrance, CA, USA).
Plant Material
The stems and leaves of mistletoe were collected in Liaoning province of China and identified by
associate Dr. Ying Jia (Department of Traditional Chinese Medicine, Shenyang Pharmaceutical
University, Shenyang, China).
Extraction and Isolation
Air-dried stems and leaves of mistletoe (10 kg) were extracted exhaustively with 95% EtOH under reflux.
The extract was concentrated under reduced pressure to get a brown syrup (1.1 kg). The syrup was
suspended in H2O and partitioned with petroleum ether, EtOAc, and n-BuOH, successively. The EtOAc
extract was concentrated to dryness (105 g), and was subjected to silica gel column with a gradient of
CH2Cl2-MeOH (100:0-0:100) to afford 18 fractions (F1-F18). F5 (CH2Cl2-MeOH 100:3) and F8
(CH2Cl2-MeOH 100:7) were further purified using a Sephadex LH-20 column to yield compounds 4 (17
mg) and 5 (20 mg), respectively. The n-BuOH extract (240 g) was eluted over polyamide column with
water, 25% EtOH, 50% EtOH, 75% EtOH and 95% EtOH, consecutively. The 75% EtOH eluate was
concentrated to dryness (30 g), and was subjected to silica gel column with a gradient of CH2Cl2-MeOH
(100:0-0:100) to afford 16 fractions (F1-F16). F7 (CH2Cl2-MeOH 100:5) was chromatographed on a
silica gel column using a gradient mixture of CH2Cl2-MeOH (100:0-0:100). Subfraction (CH2Cl2-MeOH
10:1) further purified by passage through a Sephadex LH-20 column to yield compound 2 (25 mg). F9
(CH2Cl2-MeOH 100:7) yielded compound 3 (150 mg) after recrystallization from MeOH, respectively.
F13 (CH2Cl2-MeOH 100:30) was subjected to chromatography on a silica gel column using a gradient
mixture of CH2Cl2-MeOH (100:0-0:100). Subfractions (CH2Cl2-MeOH 100:30) from F13 were further
purified using a Sephadex LH-20 column to yield compound 1 (17 mg).
Rhamnazin-3-Ο-β-D-(6′′-β-hydroxy-β-methylglutaryl)-β-D-glucoside-4′-Ο-β-D-glucoside
yellow powder; [α]2D0 -72.8 (c 0.16, MeOH); UV (MeOH, λmax, nm): 205, 251, 267, 346; IR banks (KBr)
max (cm-1): 3420, 2924, 1729, 1654, 1600, 1071, 863, 802 and 620; 1H and 13C NMR data (methanol-d4,
(1):
a
v
600 and 150 MHz, respectively) provided in Table 1. HR-ESI-MS (positive ion mode) m/z 799.2295
[M+H]+ (calcd. for C35H42O21, 799.2297). The fragments of positive ESI-MS at m/z 637.1769
[M-C6H11O5+H]+, 493.1346 [M-C6H11O5-C6H9O4+3H]+, 331.0821 [M-C6H11O5 -C12H19O9+3H]+.

HETEROCYCLES, Vol. 89, No. 6, 2014
1461
Acid Hydrolysis of Compound 1
The sample (1.0 mg) was refluxed with 1 N HCl (2.0 mL) for 5 h and then neutralized with BaCO3. The
precipitate was filtered off and the filtrate was used for detection. The sugar components were identified
as glucose by TLC over silica gel (EtOAc/EtOH/H2O/AcOH, 6:4:1:1) in comparison with authentic
sugars. The mixtures were further purified using a Sephadex LH-20 column. The specific rotation of
glucose revealed that it was a D-sugar.
Antioxidant Activity Determination
The potential antioxidant activity of these compounds was assessed on the basis of the scavenging
activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. Different concentrations (5-500
μg/mL in MeOH) of test samples were prepared. The reaction mixtures consisting of 100 μL different
concentratons of test samples and 100 μL of 100 μg/mL DPPH in methanol were fixed in quartz cuvettes
and incubated for 30 min. The absorbance (A) was measured at 515 nm. The antioxidant activity was
obtained from the following equation: Antioxidant activity (%) = [(Acontrol – Asample)/Acontrol] × 100%. The
percent antioxidant activity was plotted against concentration. The antioxidant activity of the compounds
was expressed as EC50, which was defined as the concentration in μmol/mL. The measurement was
performed in triplicate. Vitamin C was used as positive control.
ACKNOWLEDGMENT
This work was financially supported by the National Natural Science Foundation of China (Grant No.
30901967) and National Natural Science Fundation of Liaoning Province (No. 2013020223).
REFERENCES
1. H. Zwierzina, L. Bergmann, H. Fiebig, S. Aamdal, P. Schoffski, K. Witthohn, and H. Lentzen, Eur. J.
Cancer, 2011, 47, 1450.
2. P. O. Osadebe and E. O. Omeje, J. Ethnopharm., 2009, 126, 287.
3. G. S. Kienle, A. Glockmann, M. Schink, and H. Kiene, J. Exp. Clin. Cancer Res., 2009, 28, 79.
4. X. M. Piao, W. F. Chu, and G. F. Qiao, J. Harbin Med. Univ., 2006, 40, 20.
5. W. F. Chu, G. F. Qiao, Y. L. Bai, Z. W. Pan, G. Y. Li, X. M. Piao, L. W. Wu, Y. J. Lu, and B. F. Yang,
Phytother. Res., 2008, 22, 134.
6. R. J. Li, G. E. Yang, H. J. Bai, Q. Zhang, J. K. Li, Q. S. Li, and Z. M. Zhang, Chem. Nat. Comp., 2012,

1462
HETEROCYCLES, Vol. 89, No. 6, 2014
5, 761.
7. X. Yao, G. S. Zhou, Y. P. Tang, S. Guo, C. Jin, Y. Qin, D. W. Qian, and J. A. Duan, Nat. Prod. Res.
Dev., 2012, 24, 1377.
8. M. Díaz, A. González, I. Castro-Gamboa, D. Gonzalez, and C. Rossini, Carbohydr. Res., 2008, 343,
2699.
9. G. L. Boyer and S. S. Dougherty, Phytochemistry, 1988, 27, 1521.
10. L. V. Alegrio, R. B. Filho, and O. R. Gottlieb, Phytochemistry, 1989, 28, 2359.
11. J. B. He, G. M. Liu, and Y. X. Cheng, Chin. Tradit. Herb. Drugs, 2011, 42, 1905.