Four new flavonol glycosides from the leaves of Ginkgo biloba

Article abstract of DOI:10.1080/14786419.2019.1684282

Four new flavonol glycosides, 5, 7, 5′-trihydroxy-3′, 4′-dimethoxyflavonol-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (1), quercetin 3-O-(6-trans-feruloyl)-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside (2), kaempferol 3-O-(6-trans-caffeoyl)-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside (3), myricetin 3-O-(6-trans-p-coumaroyl)-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside (4), together with nine known flavonoids and two known lignans, were isolated from the leaves of Ginkgo biloba. Their structures were determined by extensive spectroscopic analyses. Their cardioprotective effects against H₂O₂-induced apoptosis in H9c2 cells were also evaluated. The flavonol glycosides had stronger activity than the acylated flavonol glycosides at the concentration of 50 μM.

Full text of DOI:10.1080/14786419.2019.1684282

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Four new flavonol glycosides from the leaves of
Ginkgo biloba
Yongxiang Wang, Xue Xie, Lina Liu, Hongda Zhang, Fuyong Ni, Jianhui Wen,
Yun Wu, Zhenzhong Wang & Wei Xiao
To cite this article: Yongxiang Wang, Xue Xie, Lina Liu, Hongda Zhang, Fuyong Ni, Jianhui Wen,
Yun Wu, Zhenzhong Wang & Wei Xiao (2019): Four new flavonol glycosides from the leaves of
Ginkgoꢀbiloba, Natural Product Research, DOI: 10.1080/14786419.2019.1684282
To link to this article: https://doi.org/10.1080/14786419.2019.1684282
View supplementary material
Published online: 04 Nov 2019.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=gnpl20

NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1684282
Four new flavonol glycosides from the leaves of
Ginkgo biloba
a,b,c
a,b,c
a,b,c
a,b,c
Yongxiang Wang , Xue Xie , Lina Liu , Hongda Zhang , Fuyong
a,b,c
a,b,c
a,b,c
a,b,c
a,b,c
Ni , Jianhui Wen , Yun Wu , Zhenzhong Wang
and Wei Xiao
a
b
Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, People’s Republic of China; State Key
Laboratory of New-Tech for Chinese Medicine Pharmaceutical Process, Lianyungang, People’s
c
Republic of China; Modern Chinese Medicine Innovation Cluster and Digital Pharmaceutical
Technology Platform, Lianyungang, People’s Republic of China
ABSTRACT
ARTICLE HISTORY
Received 2 September 2019
Accepted 20 October 2019
0
0
0
Four new flavonol glycosides, 5, 7, 5 -trihydroxy-3 , 4 -dimethoxy-
flavonol-3-O-a-L-rhamnopyranosyl-(1!6)-b-D-glucopyranoside (1),
quercetin
3-O-(6-trans-feruloyl)-b-D-glucopyranosyl-(1!2)-a-L-
KEYWORDS
Ginkgo biloba;
cardioprotective activity;
acylated flavonol glycoside
rhamnopyranoside (2), kaempferol 3-O-(6-trans-caffeoyl)-b-D-
glucopyranosyl-(1!2)-a-L-rhamnopyranoside (3), myricetin
3
-O-(6-trans-p-coumaroyl)-b-D-glucopyranosyl-(1!2)-a-L-rhamno-
pyranoside (4), together with nine known flavonoids and two
known lignans, were isolated from the leaves of Ginkgo biloba.
Their structures were determined by extensive spectroscopic anal-
2 2
yses. Their cardioprotective effects against H O -induced apoptosis
in H9c2 cells were also evaluated. The flavonol glycosides
had stronger activity than the acylated flavonol glycosides at the
concentration of 50mM.
1. Introduction
The dried leaves of Ginkgo biloba L. (Ginkgoaceae) have been used as herbal remedies
for centuries in China, and now their extracts are one of the most widely used herbal
products and/or dietary supplements in the world (Lin et al. 2008). The Ginkgo leaves
CONTACT Wei Xiao
xw_kanion@163.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1684282.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
Y. WANG ET AL.
contain large numbers of active compounds, the most important of which are flavo-
noids and terpene trilactones. The extract of Ginkgo leaves is standardised for these
two components (flavonol glycosides 24% and terpene trilactones 6%) (Mohanta et al.
2014). Active constituents in Ginkgo extract improve blood circulation, discourage clot
formation, reinforce the walls of the capillaries, and protect nerve cells from harm
when deprived of oxygen (Singh et al. 2008). Both in vitro and in vivo studies have
demonstrated the protective effects of Ginkgo leaves extract on the response to oxida-
tive stress (Varga et al. 1999).
In our investigation of constituents from Ginkgo biloba, we obtained thirteen flavo-
noids and two lignans. Compounds 1, 2, 3 and 4 were new compounds, and com-
pound 5 was previously mentioned (Xie et al. 2006), but neither the isolation method
nor the structural data was included. In this paper, we report the isolation and the
structural elucidation of compounds 1, 2, 3, 4 and 5, as well as their protective effects
on H O -induced H9c2 cells with the other ten compounds.
2
2
2. Results and discussion
Compound 1 was obtained as a pale yellow amorphous powder. Its molecular formula
was suggested as C H O from the basis of negative HR-ESI-MS (found m/z
2
9 34 17
–
þ
6
53.1733 [M－H] ) and positive HR-ESI-MS (found m/z 677.1704 [M＋Na] ) in combin-
ation with its NMR data. The H NMR and C NMR spectrum of 1 showed the pres-
ence of a flavone skeleton and two sugar moieties. The H NMR spectrum showed
1
13
1
one hydroxyl group at d 12.51 (1H, s, 5-OH), meta-coupled signals of A ring at d 6.21
(
1H, d, J ¼ 1.8 Hz, H-6) and 6.40 (1H, d, J ¼ 1.8 Hz, H-8) and two meta-coupled aromatic
0
0
protons at d 7.45 (1H, d, J ¼ 2.0 Hz, H-6 ) and 7.12 (1H, d, J ¼ 2.0 Hz, H-2 ), suggested it
0
0
0
to be a 3 , 4 , 5 -trisubstituted benzene in the B ring. It also indicated the presence of
0
0
two methoxyl signals at d 3.83 (3H, s, 4 -OCH ) and 3.76 (3H, brs, 5 -OCH ). Moreover,
3
3
0
0
the spectrum exhibited two anomeric protons at d 5.52 (1H, d, J ¼ 7.4 Hz, H-1 ) and d
000
4
.45 (1H, d, J ¼ 0.8 Hz, H-1 ) and a methyl at d 0.98 (3H, d, J ¼ 6.2 Hz), suggesting a
1 1
b-glucosyl and an a-rhamnosyl moieties. The H- H COSY and TOCSY experiments per-
mitted the full assignment of the sugar protons. The sugar moieties were detected as
13
D-glucose and L-rhamnose after acid hydrolysis and TLC analysis. The C NMR spec-
trum displayed signals for 29 carbons including flavonol disaccharide glycoside with
two methoxy groups in aromatic ring (d 56.3, 60.5). In the HMBC spectrum, the long-
0
0
rang correlations from d 5.52 (1H, d, J ¼ 7.4 Hz, H-1 ) to d 134.1 (C-3), from d 3.72 (1H,
0
0
00
000
00
000
m, H-6 a) and 3.39 (1H, m, H-6 b) to d 101.3 (C-1 ) confirmed the C-1 (glc) and C-1
00
(
rha) were attached to C-3 of aglycone and C-6 (glc), respectively. The HMBC correla-
0
0
tions between d 7.45 (1H, d, J ¼ 2.0 Hz, H-2 ) and d 156.1 (C-2), 110.2 (C-6 ), 139.1
0
0
0
0
(
C-4 ); d 7.12 (1H, d, J ¼ 2.0 Hz, H-6 ) and d 156.1 (C-2), 139.1 (C-4 ), 105.7 (C-2 );
0
0
0
methoxyl proton d 3.83 (3H, s, 3 -OCH ) and d 153.0 (C-3 ), d 3.76 (3H, s, 4 -OCH ) and
3
3
0
0
0
0
d 139.1 (C-4 ) were observed, indicating the presence of a 3 , 4 -dimethoxy-5 -hydroxy-
benzene of the B ring. Based on the evidence above, compound 1 was deduced
as 5, 7, 5 -trihydroxy-3 , 40-dimethoxyflavonol-3-O-a-L-rhamnopyranosyl-(1!6)-
0
0
b-D-glucopyranoside (Figure 1).

NATURAL PRODUCT RESEARCH
3
Figure 1. Structure of compounds 1–15.
Compound 2 was obtained as a pale yellow amorphous powder. Its molecular for-
mula was suggested as C H O from the basis of negative HR-ESI-MS (found m/z
3
7 38 19
–
þ
1
7
85.1944 [M－ H] ) and positive HR-ESI-MS (found m/z 809.1912 [M＋ Na] ). The H
NMR spectrum showed one hydroxyl group at d 12.67 (1H, s, 5-OH), two broad singlet
signals of A ring at d 6.18 (1H, s, H-6) and 6.33 (1H, s, H-8), as well as an ABX system
0
0
at d 6.90 (1H, d, J ¼ 8.4 Hz, H-5 ), 7.26 (1H, dd, J ¼ 8.4, 2.0 Hz, H-6 ) and 7.38 (H, d,
0
J ¼ 2.0 Hz, H-2 ) of B ring, revealed the existence of a quercetin moiety. Moreover, the
0
000
spectrum revealed signals of a methoxyl group at d 3.75 (3H, s, 6 -OCH ), two olefinic
3
0000
0000
doublets at d 6.35 (1H, d, J ¼ 15.9 Hz, H-2 ) and 7.45 (1H, d, J ¼ 15.9 Hz, H-3 ), a
0
000
second ABX system at d 6.72 (1H, d, J ¼ 8.2 Hz, H-8 ), 6.99 (1H, dd, J ¼ 8.2, 1.7 Hz, H-
0
000
0000
9
) and 7.21 (1H, d, J ¼ 1.7 Hz, H-5 ) suggesting a substitution by an organic acid
derivative of trans-ferulic acid. It also indicated the spectrum exhibited two anomeric
000
00
protons at d 4.29 (1H, d, J ¼ 7.8 Hz, H-1 ) and d 5.52 (1H, s, H-1 ) and a methyl at d
00
0
.92 (3H, d, J ¼ 6.2 Hz, H-6 ), suggesting a b-glucosyl and an a-rhamnosyl moieties. The
1
1
H- H COSY experiments permitted the full assignment of the sugar protons. The con-
figuration of sugar was confirm to be D-glucose and L-rhamnose by acid hydrolysis
and TLC analysis. In the HMBC spectrum, the long-rang correlations from d 5.52 (1H, s,
0
0
000
00
H-1 ) to d 134.7 (C-3), from d 4.29 (1H, d, J ¼ 7.8 Hz, H-1 ) to d 82.2 (C-2 ) confirmed
0
0
000
00
the C-1 (rha) and C-1 (glc) were attached to C-3 of aglycone and C-2 (rha), and the
esterification of the glucose unit was confirmed by the correlations observed between
0
000
000
d 167.9 (C-1 ) and both d 4.04 (1H, d, J ¼ 11.8 Hz, H-6a ) and 4.22 (1H, dd, J ¼ 11.8,
000
3
.8 Hz, H-6b ). Analysis of the ROESY spectrum showed a correlation between the
0000
0000
methoxy protons d 3.75 (3H, s, 6 -OCH ) and proton d 7.21 (1H, d, J ¼ 1.7 Hz, H-5 ),
3
0
0000
and allowed us to locate the methoxy group at C-6 . Based on the evidence above,

4
Y. WANG ET AL.
compound 2 was deduced as quercetin 3-O-(6-trans-feruloyl)-b-D-glucopyranosyl-
(
1!2)-a-L-rhamnopyranoside.
Compound 3 was obtained as a pale yellow amorphous powder. Its molecular for-
mula was suggested as C H O from the basis of negative HR-ESI-MS (found m/z
3
6 36 18
–
þ
1
7
55.1897 [M－ H] ) and positive HR-ESI-MS (found m/z 779.1835 [M＋ Na] ). The H
NMR spectrum had two broad singlet signals at d 6.19 (1H, s, H-6) and 6.37 (1H, s, H-
0
0
0
0
8
) of A ring, and an AA BB system at d 7.38 (2H, d, J ¼ 8.8 Hz, H-2 , 6 ), 6.93 (2H, d,
0
0
J ¼ 8.8 Hz, H-3 , 5 ) of B ring, revealed the existence of a kaempferol moiety. Moreover,
the spectrum revealed signals of two olefinic doublets at d 6.11 (1H, d, J ¼ 15.9 Hz, H-
0
0
000
000
0000
0000
2
8
) and 7.39 (1H, d, J ¼ 15.9 Hz, H-3 ), a ABX system at d 6.69 (1H, d, J ¼ 8.2 Hz, H-
0000
), 6.87 (1H,dd, J ¼ 8.2, 1.7 Hz, H-9 ) and 6.96 (1H, d, J ¼ 1.7 Hz, H-5 ) suggesting a
1
13
substitution by a trans-caffeic acid. Compared H and C NMR spectroscopic data of 3
with those of 2 revealed that the structures of 3 had the same sugar moieties as 2, D-
glucose and L-rhamnose were identified by TLC upon acid hydrolysis of 3. In the
HMBC spectrum, the kaempferol was glycosylated at position 3, as observed by the
0
0
long-rang correlations between d 5.60 (1H, s, H-1 ) and d 134.8 (C-3); and the esterifi-
cation of the glucose was confirmed by the correlations between d 167.9 (C-1 ) and
d 4.18 (2H, m, H-6 ). Based on the evidence above, compound 3 was deduced as
kaempferol 3-O-(6-trans-caffeoyl)-b-D-glucopyranosyl-(1!2)-a-L-rhamnopyranoside.
Compound 4 was obtained as a pale yellow amorphous powder. Its molecular for-
mula was suggested as C H O from the basis of negative HR-ESI-MS (found m/z
0
000
000
3
6 36 19
–
þ
1
7
71.1788 [M－ H] ) and positive HR-ESI-MS (found m/z 795.1759 [M＋ Na] ). The H
NMR spectrum showed one hydroxyl group at d 12.67 (1H, s, 5-OH), two broad singlet
signals of A ring at d 6.17 (1H, s, J ¼ 1.7 Hz, H-6) and 6.33 (1H, s, J ¼ 1.7 Hz, H-8), as
0
0
well as a singlet at d 6.96 (2H, s, H-2 , 6 ) of B ring, revealed the existence of a myrice-
tin moiety. Moreover, the spectrum revealed signals of two doublets at d 6.27 (1H, d,
0
000
0000
J ¼ 15.9 Hz, H-2 ) and 7.46 (1H, d, J ¼ 15.9 Hz, H-3 ) which identified trans olefinic
0
0
0000
0000
double bond protons, and an AA BB system at d 6.72 (2H, d, J ¼ 8.6 Hz, H-6 , 8 )
0000
0000
and 7.44 (2H, d, J ¼ 8.6 Hz, H-5 , 9 ), which confirmed the presence of the trans-p-
1
13
coumaroyl subunit. Compared H NMR, C NMR and HMBC spectroscopic data of 4
with those of 2 revealed that the structures of 4 had the same sugar components as
2
, and D-glucose and L-rhamnose were identified by TLC upon acid hydrolysis of 4. In
the HMBC spectrum, the myricetin was glycosylated at position 3, as observed by the
0
0
long-rang correlations between d 5.46 (1H, s, H-1 ) and d 133.3 (C-3); a cross peak
0
00
000
000
between d 165.3 (C-1 ) and both d 3.96 (1H, d, J ¼ 11.5 Hz, H-6a ) and 4.22 (1H, d,
0
J ¼ 11.5, 3.7 Hz, H-6b ) established the linkage points between the two sugar and the
trans-p-coumaroyl moieties. On the basis of these data, compound 4 was elucidated as
myricetin 3-O-(6-trans-p-coumaroyl)-b-D-glucopyranosyl-(1!2)-a-L-rhamnopyranoside.
Compound 5 was obtained as a yellow amorphous powder. Its molecular formula
was suggested as C H O from the basis of negative HR-ESI-MS (found m/z
3
6 36 17
–
þ
1
7
39.1889 [M－ H] ) and positive HR-ESI-MS (found m/z 763.1856 [M＋ Na] ). The H
NMR spectrum showed the characteristic proton signals at d 6.35 (1H, d, J ¼ 1.6 Hz, H-
0
0
8
), 6.19 (1H, d, J ¼ 1.6 Hz, H-6), 7.66 (2H, d, J ¼ 8.7 Hz, H-2 , 6 ), 6.91 (2H, d, J ¼ 8.7 Hz,
0
0
H-3 , 5 ), and 12.56 (1H, brs, 5-OH) revealed the existence of a kaempferol moiety.
Moreover, the spectrum exhibited two doublets at d 5.34 (1H, d, H-2 ) and 6.39 (1H,
0
000

NATURAL PRODUCT RESEARCH
5
0
000
d, H-3 ) with coupling constants of J ¼ 12.8 Hz which assigned cis olefinic double
bond protons, and the other caused by the four aromatic ring proton signals at d 7.48
0
000
0000
0000
0000
0
0
(
2H, d, J ¼ 8.7 Hz, H-5 , 9 ) and 6.69 (2H, d, J ¼ 8.7 Hz, H-6 , 8 ) (AA BB ), which
1
confirmed the presence of the cis-p-coumaroyl subunit (Lee et al. 2013). Compared H
1
3
NMR, C NMR and HMBC spectroscopic data of 5 with those of 2 revealed that the
structures of 5 had the same sugar components as 2, D-glucose and L-rhamnose were
identified by TLC upon acid hydrolysis of 5. In the HMBC spectrum, a cross peak
00
between d 5.61 (1H, d, J ¼ 1.0 Hz, H-1 ) and d 137.4 (C-3), and between d 4.28 (2H, m,
0
00
0000
H-6 ) and d 167.9 (C-1 ) established the linkage points between the two sugar and
the p-cis-coumaroyl moieties. On the basis of these data, compound 5 was elucidated
as kaempferol 3-O-(6-cis-p-coumaroyl)-b-D-glucopyranosyl-(1!2)-a-L-rhamnopyrano-
side. This compound was previously reported (Xie et al. 2006) but neither the isolation
method nor the structural data was included.
Additionally, the known compounds (6–15) were identified by comparing the
spectroscopic data with those reported in the literature to be rutin (6) (Wang et al.
1
999), kaempferol 3-O-(6-p-coumaroyl)-b-D-glucopyranosyl-(1!2)-a-L-rhamnopyrano-
side (7) (Markham et al. 1992), quercetin 3-O-a-(6-p-coumaroyl)-b-D-glucopyranosyl-
(
(
1!2)-a-L-rhamnopyranoside (8) (Markham et al. 1992), syringetin-3-O-rutnoside (9)
Victoire et al. 1988), isorhamnetin-3-O-rutinoside (10) (Victoire et al. 1988), kaemp-
ferol-3-O-b-D-glucopyranosyl-(1!2)-a-L-rhamnopyranoside (11) (Hasler et al. 1992),
dihydrodehydrodiconiferyl alcohol-4-O-b-D-glucopyranoside (12) (Matsuda et al.
0
1
996), lariciresinol-4 -O-b-D-glucoside (13) (Sugiyama and Kikuchi 1993), quercetin-3-
O-b-D-glucopyranosyl-(1!2)-a-L-rhamnopyranoside (14) (Hasler et al. 1992), kaemp-
ferol-3-O-rutinoside (15) (Markham et al. 1978). Among them, compounds 9 and 10
were isolated from the Ginkgo genus for the first time.
All the isolates were evaluated for their antioxidative effects against H O -induced
2
2
cytotoxicity in cultured H9c2 cells. Among them, the inhibition rates of compound 9
was 50.34%, compounds 10 and 13 were more than 30%. The flavonol glycosides had
stronger activity than the acylated flavonol glycosides at the concentration of 50 mM.
None of the compounds showed a significant cytotoxicity on H9c2 cells at the
concentration of 50 lM.
3. Conclusions
Thirteen flavonoids and two lignans were obtained, compounds 1, 2, 3 and 4 were
new flavonoid glycosides, and the NMR data of compound 5 was unprecedented and
fully assigned for the first time. All the isolates were evaluated for their antioxidative
effects against H O -induced apoptosis in H9c2 cells. All the isolates were evaluated
2
2
for their antioxidative effects against H O -induced cytotoxicity in cultured H9c2 cells.
2
2
The flavonol glycosides had stronger activity than the acylated flavonol glycosides
at the concentration of 50 mM
Disclosure statement
No potential conflict of interest was reported by the authors.

6
Y. WANG ET AL.
Funding
This work was supported by the National Science and Technology Major Project ‘Key New Drug
Creation and Manufacturing Program’ [grant number 2013ZX09402203].
References
Hasler A, Gross GA, Meier B, Sticher O. 1992. Complex flavonol glycosides from the leaves of
Ginkgo biloba. Phytochemistry. 31(4):1391–1394.
Lee YG, Cho JY, Kim CM, Lee SH, Kim WS, Jeon TI, Park KH, Moon JH. 2013. Coumaroyl quinic
acid derivatives and flavonoids from immature pear (Pyrus pyrifolianakai) fruit. Food Sci
Biotechnol. 22(3):803–810.
Lin LZ, Chen P, Ozcan M, Harnly JM. 2008. Chromatographic profiles and identification of new
phenolic components of Ginkgo biloba leaves and selected products. J Agric Food Chem.
56(15):6671–6679.
Markham KR, Geiger H, Jaggy H. 1992. Kaempferol-3-O-glucosyl(1-2)rhamnoside from Ginkgo
biloba and reappraisal of other gluco(1-2, 1-3, and 1-4)rhamnoside structures. Phytochemistry.
31(3):1009–1011.
Markham KR, Ternai B, Stanley R, Geiger H, Mabry TJ. 1978. Carbon-13NMR studies of flavo-
noids-III: naturally occurring flavonoid glycosides and their acylated derivatives. Tetrahedron.
34(9):1389–1397.
Matsuda N, Sato H, Yaoita Y, Kikuchi M. 1996. Isolation and absolute structures of the neolignan
glycosides with the enantiometric aglycones from the leaves of Viburnum awabuki K. KOCH.
Chem Pharm Bull. 44(5):1122–1123.
Mohanta TK, Tamboli Y, Zubaidha PK. 2014. Phytochemical and medicinal importance of Ginkgo
biloba L. Nat Prod Res. 28(10):746–752.
Singh B, Kaur P, Gopichand Singh RD, Ahuja PS. 2008. Biology and chemistry of Ginkgo biloba.
Fitoterapia. 79:401–418.
Sugiyama M, Kikuchi M. 1993. Characterization of lariciresinol glucosides from Osmanthus
asiaticus. Heterocycles. 36:117–121.
Varga E, Bodi A, Ferdinandy P, Droy-Lefaix MT, Blasig IE, Tosaki A. 1999. The protective effect
of EGb 761 in isolated ischemic/reperfused rat hearts: a link between cardiac function and
nitric oxide production. J Cardiovasc Pharmacol. 34(5):711–717.
Victoire C, Haag-Berrurier M, Lobstein-Guth A, Balz J, Anton R. 1988. Isolation of flavonol
glycosides from Ginkgo biloba leaves. Planta Med. 54(03):245–247.
Wang M, Kikuzaki H, Csiszar K, Boyd CD, Maunakea A, Fong SFT, Ghai G, Rosen RT, Nakatani N,
Ho C-T. 1999. Novel trisaccharide fatty acid ester identified from the fruits of Morinda citrifolia
(noni). J Agric Food Chem. 47(12):4880–4882.
Xie P, Chen S, Liang YZ, Wang X, Tian R, Upton R. 2006. Chromatographic fingerprint analysis-a
rational approach for quality assessment of traditional Chinese herbal medicine. J Chromatogr
A. 1112(1–2):171–180.