Characterization and antiglycation activity of phenolic constituents from Viscum album (European Mistletoe)

Article abstract of DOI:10.1248/cpb.58.980

4′-O-[β-D-Apiosyl(1→2)]-β-D-glucosyl] -5-hydroxyl-7-O-sinapylflavanone (1), 3-(4-acetoxy-3,5-dimethoxy)-phenyl-2E- propenyl-β-D-glucopyranoside (2), 3-(4-hydroxy-3,5-dimethoxy)-phenyl-2E- propenyl-β-D-glucopyranoside (3), 5,7-dimethoxy-4′-O-β-D- glucopyra

Full text of DOI:10.1248/cpb.58.980

9
80
Note
Chem. Pharm. Bull. 58(7) 980—982 (2010)
Vol. 58, No. 7
Characterization and Antiglycation Activity of Phenolic Constituents
from Viscum album (European Mistletoe)
,
a
a
a
a
a
M. Iqbal CHOUDHARY,* Saima MAHER, Afshan BEGUM, Ahmed ABBASKHAN, Sajjad ALI,
a
b
,a
Ambreen KHAN, Shafique-ur-REHMAN, and Atta-ur-RAHMAN*
a
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi;
b
Karachi-75270, Pakistan: and Department of Botany, University of Azad Jammu and Kashmir; Muzafarabad-13100,
Pakistan. Received February 2, 2010; accepted February 4, 2010; published online April 2, 2010
4
؅-O-[b-D-Apiosyl(1→2)]-b-D-glucosyl]-5-hydroxyl-7-O-sinapylflavanone (1), 3-(4-acetoxy-3,5-dimethoxy)-
phenyl-2E-propenyl-b-D-glucopyranoside (2), 3-(4-hydroxy-3,5-dimethoxy)-phenyl-2E-propenyl-b-D-glucopyra-
noside (3), 5,7-dimethoxy-4؅-O-b-D-glucopyranoside flavanone (4), 4؅,5-dimethoxy-7-hydroxy flavanone (5), and
,7-dimethoxy-4؅-hydroxy flavanone (6), were isolated from the organic extracts of Viscum album L. (European
5
Mistletoe). These compounds were studied for their anti-glycation and antioxidant properties. The structures of
new compounds 1 and 2 were deduced on the basis of spectroscopic evidence.
Key words Viscum album; European Mistletoe; Loranthaceace; anti-glycation activity; antioxidant activity; phenylpropanoid
1
13
Viscum album L. (Loranthaceace), commonly known as Table 1. H- and C-NMR Chemical Shift Data for Compounds 1 and 2
European Mistletoe, is a bi-perennial shrub, widely distrib-
Compound 1
Compound 2
uted in tropical and sub-tropical regions of Africa, Asia and
1,2)
Europe. It is commonly found as a semi parasite on trees.
Some species of the genus Viscum are used in folk medicines
for the treatment of diabetes, jaundice, indigestion, common
dH
dC
dH
dC
1
2
3
—
—
1
2
3
—
135.8
105.4
154.3
3)
5.23 (dd, Jϭ12.8, 3.1 Hz)
2.64 (dd, Jϭ17.8, 3.1 Hz)
79.1
6.74 (s)
—
fever and asthma. In Pakistan, it is found in Neelam valley
Azad Kahsmir) as a hemi-parasite on the trees of Juglans
(
2
.86 (dd, Jϭ17.8, 12.8, Hz) 44.0
regia (Walnut).
4
5
6
7
8
9
0
1Ј
—
197.6
168.9
94.8
4
5
6
7
8
9
—
135.3
154.4
105.4
110.5
A number of biological activities of mistletoe, including
immunostimulatory and antitumor activity, have been re-
—
—
6.01 (d, Jϭ2.3 Hz)
6.75 (s)
4)
—
164.3
95.8
6.57 (d, Jϭ15.0 Hz)
ported. Previous investigation resulted in the isolation of
lectins, viscotoxins, alkaloids, amines, flavonoids, acids and
5.97 (d, Jϭ2.3 Hz)
6.36 (dt, Jϭ15.0, 5.54 Hz) 130.0
—
162.6
107.1
128.7
126.3
117.6
158.9
117.6
4.22 (Jϭ2.0 Hz)
63.4
105.3
75.6
77.7
78.4
71.4
62.4
180
5,6)
terpenoids. Anti-glycation potential of the methanolic ex-
1
—
1Ј 4.86 (d, Jϭ7.2 Hz)
2Ј 3.38 (t, Jϭ7.2 Hz)
3Ј 3.38 (t, Jϭ7.8 Hz)
4Ј 3.41 (dd, Jϭ7.0, 4.3 Hz)
5Ј 3.44 (m)
7)
tract of V. album was also reported earlier by Gray and Flat.
—
2
3
4
5
6
Ј
Ј
Ј
Ј
Ј
7.31 (d, Jϭ8.6 Hz)
7.02 (d, Jϭ8.7 Hz)
—
During the biological screening of medicinal herbs of Pak-
istani origin, the crude extract of V. album showed a signifi-
cant anti-glycation activity (Table 1). Non-enzymatic glyca-
tion of proteins is a process that impairs the functioning of
biomolecules and leads to a variety of endocrine disorders.
Free radicals also contributed in the formation of advanced
7.31 (d, Jϭ8.6 Hz)
7.02 (d, Jϭ8.6 Hz)
6Ј 3.82 (Jϭ7.5, 11.5 Hz)
126.3 OAc 1.88 (s)
(OCϭOCH3)
128.6
30.7
1
Љ
—
8)
2Љ
6.82 (s)
106.2
glycation end products (AGEs). Eventually this may lead to
oxidative stress and a variety of degenerative diseases, such
3
4
Љ
Љ
—
149.4
—
133.9
9)
as cancer, diabetes, inflammation and aging. The elimina-
tion or inactivation of reactive oxygen species (ROS) is con-
sidered to be an instrumental approach to reduce the risk of
5Љ
6Љ
—
149.4
6.82 (S)
106.8
7
Љ
7.59 (d, Jϭ15.8 Hz)
6.31( d, Jϭ15.8 Hz)
—
147.9
10)
8Љ
115.5
these diseases.
9
Љ
162.8
The present work resulted in the isolation of compounds
OCH3 3.82 (s)
56.5
1
—6, which were found to posses anti-glycation activity,
Glucose
whereas compounds 2 and 5 exhibited anti-oxidant activities.
Among the recently isolated compounds flavanone glycoside
C-1ٞ 4.93 (d, Jϭ7.9 Hz)
C-2ٞ 3.64 (dd, Jϭ9.2, 7.5 Hz)
C-3ٞ 3.99 (t, Jϭ9.2 Hz)
C-4ٞ 3.84 (dd, Jϭ9.2, 6.8 Hz)
C-5ٞ 3.40 (m)
100.5
78.1
77.7
71.6
71.4
62.3
(1) and phenyl propoanoid (2), were found to be new. The
structures of the isolated compounds were elucidated mainly
with the help of NMR spectroscopic techniques.
C-6ٞ 3.67 (dd, Jϭ14.3, 7.3 Hz)
Apiose
C-1ЉЉ 5.54 (d, Jϭ1.4 Hz)
C-2ЉЉ 3.91 (d, Jϭ1.4 Hz)
110.3
75.8
79.0
78.8
67.5
Results and Discussion
The air dried powdered plant material was extracted with
C-3ЉЉ
—
80% aqueous methanol. The flavanone 1 and phenyl pro-
C-4ЉЉ 4.24 (d, Jϭ11.5 Hz)
C-5ЉЉ 4.39 (d, Jϭ11.3 Hz)
panoid 2 were isolated from the n-BuOH and EtOAc extracts
of the plant, respectively.
1
13
H-NMR (400 MHz in CD OD), C-NMR (100 MHz in CD OD).
3
3
Compound 1 was obtained as a colorless powder. The IR
∗
To whom correspondence should be addressed. e-mail: hej@cyber.net.pk
© 2010 Pharmaceutical Society of Japan

July 2010
981
urated carbonyl (d 162.8), and two methine carbons (d
C
C
1
47.9, 115.5) indicated the presence of a sinapyl moiety. A
bathochromic shift of band II (ca. 24 nm) in the presence of
shift reagent (AlCl /HCl) indicated C-5 hydroxyl functional-
3
13)
ity. The only possible position of sinapyl moiety in ring A
at OH of C-7 was deduced from the downfield shift of C-7
14)
(
d 164.3).
C
The nature of glycosides was deduced by the hydrolysis
see Experimental). The stereochemistry at C-2 position was
(
assigned as S on the basis of the circular dichroism (CD)
spectrum, in which compound 1 exhibited a positive Cotton
1)
effect at 330 nm and a negative Cotton effect at 286 nm.
Based on above mentioned evidences, the new compound
was deduced as 4Ј-O-[b-D-apiosyl(1→2)]-b-D-glucopyra-
nosyl]-5-hydroxyl-7-O-sinapylflavanone (1).
Fig. 1. Structures of the Compounds 1—6
Compound 2 was isolated as a white amorphous powder.
The IR spectrum showed absorptions at 3385, 1735, 1660
Ϫ1
spectrum showed absorptions at 3412 (OH), 1668 (a,b-un- and 1593 cm , indicating the presence of hydroxyl group,
Ϫ1
saturated carbonyl), and 1606, 1520 cm (CϭC). The UV carbonyl ester and an aromatic ring, respectively. UV spec-
spectrum showed absorptions at 326 and 283 nm, characteris- trum showed absorption indicative of the presence of 261
tic of a flavanone skeleton and sinapyl group, respectively. (3.45), 242 (3.77), 203 (4.05), 189 (4.07). The HR-FAB-MS
ϩ
The HR-fast bombardment (FAB)-MS (ϩve) showed the (ϩve) showed [MϩH] at m/z 415.1522 corresponded to the
ϩ
[
MϩH] at m/z 773.1073, corresponding to the formula formula C H O (Calcd 415.1526). The overall spectral
1
9
27 10
C H O (Calcd 773.1079). The electron ionization-mass data of compound 2 closely resembled to a known com-
3
7
41 18
spectra (EI-MS) showed the presence of a sinapyl moiety as pound, 3-(4-hydroxy-3,5-dimethoxy)-phenyl-2E-propenyl-b-
ϩ
15)
inferred from the fragment ion C H O at m/z 207. The D-glucopyranoside (3), the only difference between the two
1
1
11
4
presence of sugar moieties (apiose and glucose) was inferred compounds being the appearance of signals for acetate
ϩ
by the of [MϪ462] at m/z 311 in HR-FAB-MS (ϩve). The (O–CϭOCH ) moiety in the NMR spectra of 2. A downfield
3
1
H-NMR spectrum of 1 showed the presence of three mutu- methyl singlet at d 1.88 indicated the presence of an acetate
H
1
3
ally coupled protons double doublet at d 2.64 (1H, Jϭ17.8, moiety, whereas a downfield signal in C-NMR spectrum at
H
3
.1 Hz) and 5.23 (1H, Jϭ12.8, 3.1 Hz), corresponding to the d 180.0, along with a methyl signal at d 30.7, further sup-
C
C
C-3 methylene and C-2 methine proton of ring C of fla- ported the presence of acetate functionality.
vanone, respectively. A 2H doublet at dH 7.31 with the cou-
The substitution of the acetate moiety at C-4 was deduced
pling constant 8.6 Hz was assigned to C-3Ј and C-5Ј aromatic from the downfield shift of C-4 (d 135.3). The above spectral
protons. Similarly, another 2H doublet at dH 7.02 with the evidence and comparison of data supported the structure
coupling constant 8.6 Hz was assigned to C-2Ј and C-6Ј mag- of compound 2 as 3-(4-acetoxy-3,5-dimethoxy)-phenyl-2E-
netically equivalent aromatic protons. Two meta coupled pro- propenyl-b-D-glucopyranoside.
tons of ring A appeared at d 5.97 (1H, Jϭ2.3 Hz) and 6.01
The known compounds, isolated from V. album, were iden-
H
(
1H, Jϭ2.3 Hz), corresponding to the C-8 and C-6 protons, tified as 3-(4-hydroxy-3,5-dimethoxy)-phenyl-2E-propenyl-
1
5)
respectively. The presence of a sinapyl moiety in compound b-D-glucopyranoside.(3), 5,7-dimethoxy-4Ј-O-b-D-glu-
12)
1
was inferred from an AB doublets of the trans olefinic pro- copyranoside flavanone (4), 4Ј,5-dimethoxy-7-hydroxy fla-
16)
17)
tons at d 7.59 (1H, Jϭ15.8 Hz) and 6.31 (1H, Jϭ15.8 Hz) vanone (5), and 5,7-dimethoxy-4Ј-hydroxy flavanone (6).
H
and a two proton singlet at d 6.82, assigned to the C-2Љ and The structures of these compounds were determined by com-
H
1
13
C-6Љ protons of the phenyl ring. Two methoxy groups were paring the EI-MS, H- and C-NMR data with the reported
1
inferred from a singlet at dH 3.82 (s, 6H). The H-NMR data.
spectrum also showed signals (d 3.40—5.54) corresponding
Biological Activities The methanolic extract of V. album
potent anti-glycation activity, i.e. 72.5%
H
to sugar moieties. Two anomeric protons appeared as dou- showed
a
blets at d 5.54 (Jϭ1.4 Hz) and 4.93 (Jϭ7.9 Hz), were in (IC ϭ199.85Ϯ0.067 mM). Compounds 1—6 showed activi-
H
50
11)
agreement with those of apiose and glucose moieties. The ties against the formation of AGEs (Table 2) the antioxidant
carbon signals for sugars were also characteristic of b-D-glu- activity of these compounds was evaluated by using a super-
1
)
copyranosyl and b-D-apiofuranosyl sugars.
oxide anion scavenging assay. Compounds 2 (IC ϭ211.69Ϯ
50
The structure of compound 1 was finally deduced by de- 7.11 mM) and 5 (IC ϭ58.36Ϯ2.9 mM) were found to be ac-
tailed interpretation of 2-D NMR data. In the heteronuclear tive in this assay. The antioxidant potential of compound 5
5
0
multiple bond correlation (HMBC) spectrum, the signal at was determined to be greater than that of standard (Table 3).
3
d
4.93 (C-1ٞ) was J correlated with dC 158.9 (C-4Ј), n-Propylgallate was used as a standard reference (IC ϭ
H
50
3
whereas the apiose anomeric proton (d 5.54) showed a J 67.5Ϯ0.9 mM). In conclusion, we report here for the first
H
connectivity with C-2ٞ (d 78.1), suggesting the attachment time, anti-glycation properties of the plant phenolics, ob-
C
of apiose at C-2ٞ of glucose. This was further inferred from tained from V. album.
the downfield shift of C-2 of the glucose (dC 79.1). The
Experimental
NMR data of sugar was in agreement with those of apiose
General Methods Unless otherwise stated, the following procedures
and glucose moieties. Characteristic signals for a,b-unsat- were adopted. UV spectra were measured on a Shimadzu UV240 machine in
12)

9
82
Vol. 58, No. 7
Table 2. Anti-glycation Activity of Compounds of V. album
polyamide column chromatography and eluted with 15% MeOH–CHCl3
which yielded two major fractions (P1, 1 g and P2, 575 mg). The sub-frac-
tion P1 was further subjected to Sephadex LH-20 column chromatography
b)
Compounds/Extracts
Inhibition (%)
IC50 (mM)ϮS.E.M.
and eluted with H O–MeOH (1 : 1), to afford three fractions (7—10). Frac-
2
Methanolic extract
Ethyl acetate extract
Butanolic extract
72.5
66.5
199.8Ϯ0.1
270.7Ϯ0.4
689.4Ϯ0.5
264.5Ϯ0.9
255.4Ϯ0.5
413.9Ϯ0.5
345.6Ϯ0.8
264.5Ϯ0.7
405.8Ϯ0.8
67.5Ϯ0.9
tion 7 (15 mg) was loaded onto RHPLC (L-80, H O : MeOH, 1 : 1, 4 ml/min)
2
Ϫ4
to yield compound 1 (3 mg, 1.0ϫ10 %, t 25 min).
R
61.45
74.5
71.36
72.92
71.4
74.62
73.8
85.9
4Ј-O-[b-D-Apiosyl(1→2)]-b-D-glucosyl]-5-hydroxyl-7-O-sinapylfla-
2
5
1
2
3
4
5
6
vanone (1): Colorless powder [a]D ϩ8.4 (cϭ3.5, MeOH). UV lmax
(MeOH) nm (log e): 326 (2.75), 283 (3.04), 214 (3.33), 206 (3.35). IR (KBr)
Ϫ1
1
nmax cm : 3412 (OH), 1668, 1606, 1520, H-NMR (CD OD, 400 MHz): see
3
1
3
Table 1; C-NMR (CD OD, 100 MHz): see Table 1; EI-MS at m/z
3
ϩ
ϩ
(C H O ) 207, [MϪ462] at m/z 311 sugar moieties (apiose and glucose)
11 11 4
HR-FAB-MS (ϩve). HR-FAB-MS m/z: (Calcd for C H O : 773.1079).
3
7
41 19
a)
ϩ
Rutin
[MϩH] 773.1073.
-(4-Acetoxy-3,5-dimethoxy)-phenyl-2E-propenyl-b-D-glucopyranoside
3
2
5
a) Standard. b) Results are reported in Ϯstandard error of mean of three experi-
ments.
(2): White amorphous powder [a] ϩ65 (cϭ8.5, MeOH). UV lmax
D
(
MeOH) nm (log e): 261 (3.45), 242 (3.77), 203 (4.05), 189 (4.07). IR (KBr)
Ϫ
1
1
cm : 3385, 1735, 1660, 1593. H-NMR (400 MHz, CD OD): see Table 1;
3
1
3
C-NMR (CD OD, 100 MHz) see Table 1; HR-FAB-MS m/z: (Calcd for
Table 3. Superoxide Anion Scavenging Activity of Compounds of V.
album
3
ϩ
C H O , 415.1526). 415.1522 [MϩH] C H O .
19 27 10
19 27 10
Acid Hydrolysis of Compound 1 Compound 1 (1.3 mg) was dissolved
b)
in 5% HCl/H O (2 ml) and refluxed for 1 h. The solution was extracted with
ethyl acetate (1 mlϫ3) to afford aglycone. While the aqueous part was neu-
Compounds
Inhibition (%)
IC50 (mM)ϮS.E.M.
2
tralized by NaHCO (pH 6) and hydrolyzed sugars were identified by paper
2
5
74.75
95.40
211.7Ϯ7.0
58.5Ϯ3.0
3
chromatography using standard sugars in the solvent system EtOAc/AcOH/
1)
H O (5 : 3 : 2). The results indicated the presence of apiose and glucose.
2
a) Standard. b) Results are reported in Ϯstandard error of mean of three experi-
ments.
Assay of Anti-glycation and Superoxides Anion The anti-glycation
and superoxide Anion assays on various secondary metabolites of Viscum
18,19)
album were carried out according to established protocols.
MeOH solutions as lmax nm (log e). IR spectra were recorded as KBr discs
Acknowledgments We are grateful to the IDB (The Islamic Develop-
ment Bank, Jeddah and KSA) for financial support to M.I.C. for the research
Ϫ1
1
13
on a JASCO A-302 spectrometer and presented in cm . H- and C-NMR,
HMQC and HMBC spectra were recorded on a Bruker AV-400 spectrometer, project entitled, “Phytochemical, Pharmacological and Toxicological Studies
1
13
operating at 400 ( H) and 100 ( C) MHz in CD OD. The chemical shifts on Medicinal Plants of Sudan and Pakistan.”
3
values were reported in d (ppm), referenced with respect to the residual sol-
vent signal of CD OD and coupling constants (J) were measured is Hz. EI- References
3
MS were taken at 70 eV on a Finnigan MAT-112 or MAT-312 instrument and
major ions are presented as m/z (%). FAB-MS were measured as a glycerol
matrix on a JEOL HX110 Mass spectrometer. TLC purification was carried
out on pre-coated silica gel cards (E. Merck) and the spots were observed
first under UV (254 nm) and then sprayed with cerium(IV) sulfate reagent
and heated until coloration developed. Recycling preparative HPLC (RPH-
PLC) was used for final purification (JAI LC-908W, Japan Analytical Indus-
try Co., Ltd.) with a column YMC ODS H-80 or L-80 (YMC, Japan).
Plant Material The whole plant of Viscum album L. was collected from
walnut (Juglans regia) trees from Kundershah, Neelum Valley, Azad Kash-
mir, on the 15th of August 2002, and identified by Prof. Shafiq-ur-Rehman.
A voucher specimen (No. Azbuherb 231) was deposited in the Herbarium of
the University of Azad Jammu and Kashmir, Muzaffarabad.
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Extraction and Isolation The methanolic crude extract (45 g) was sus-
pended in dist. H O and partitioned with EtOAc (3 lϫ3) and n-BuOH
2
(3 lϫ3) successively, yielding EtOAc (1.2 g), and n-BuOH extracts (19.4 g).
The EtOAc extract was subjected to column chromatography over silica gel
and eluted with CHCl and MeOH in a gradient manner (mention different 10) Pietta P. G., J. Nat. Prod., 63, 1035—1042 (2000).
3
proportions of solvents) to afford eight fractions (E1—E8). Repeated col- 11) Hosoya T., Yun Y. S., Kunugi A., Tetrahedron, 61, 7037—7044
umn chromatography of fraction E-2 (100 mg) over silica gel using the sol-
vent system CHCl and MeOH (10%, 600 ml) yielded compounds 6 (5 mg, 12) Fukunaga T., Kajikawa I., Nishiya K., Watanabe Y., Takeya I. H.,
(2005).
3
Ϫ4
Ϫ4
1
.6ϫ10 %) and 4 (6 mg, 2.0ϫ10 %). The third fraction (E-3, 60 mg) was
Chem. Pharm. Bull., 35, 3292—3297 (1987).
again chromatographed using a silica gel and eluted with 20% MeOH–
13) Mebry T. J., Markham K. R., Thomas, M. B,. “The Systematic Identifi-
cation of Flavonoids,” Springer-Verlag, New York, 1975, pp. 33—35.
Ϫ4
CHCl (600 ml) to yield 5 (3 mg, 1.0ϫ10 %). Fractions E-4 (23 mg) and E-
3
5
(45 mg) were combined and subjected to polyamide column chromatogra- 14) Wei L., Koike K., Tatsuzaki M., Koide, A., Nikaido T., Cucurbitosides
phy and eluted with 100% CHCl , followed by gradual increase of polarity
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with MeOH. This led to five sub-fractions (1—5). Among these, sub-frac- 15) Greca M. D., Ferrara M., Fiorentino A., Monaco L., Previtera L., Phy-
tion 4 was subjected to silica gel column chromatography using 30% MeOH
tochemistry, 49, 1299—1304 (1988).
3
Ϫ4
in CHCl (300 ml) as eluent to obtain compounds 3 (10 mg, 3.33ϫ10 %) 16) Tanaka R., Matsunaga S., Sasaki T., Planta Med., 55, 570—571
3
Ϫ4
and 2 (5.5 mg, 2.5ϫ10 %).
(1989).
A part of n-BuOH fraction (10 g) was passed through a Diaion HP-20 col- 17) Lam J., Wrang P., Phytochemistry, 14, 1621—1623 (1975).
umn and eluted with 100% H O (2.7 g), H O–MeOH (1 : 1, 3.5 g), and 100%
18) Kim H. Y., Kim K. J., J. Agric. Food Chem., 51, 1586—1591 (2003).
MeOH (2.0 g). The fraction eluted with H O–MeOH (1 : 1) was subjected to 19) Candan F., J. Enzyme Inhib. Med. Chem., 18, 59—62 (2003).
2
2
2