Flavonol glycosides from Fissistigma maclurei

Article abstract of DOI:10.1080/10286020.2019.1671374

Two new flavonol glycosides, fissmacosides A (1) and B (2) along with two known flavonol glycosides, kaempferol 3-O-α-l-rhamnopyranosyl-(1→6)-β-d-galactopyranoside (3) and kaempferol 3-O-β-d-glucopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→6)-[4-(E)-feruloyl]-β-d-galactopyranoside (4) were isolated from the methanol extract of the leaves of Fissistigma maclurei Merr. Their structures were determined on the basis of extensive spectroscopic methods, including 1D-, 2D-NMR, and MS data.

Full text of DOI:10.1080/10286020.2019.1671374

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Flavonol glycosides from Fissistigma maclurei
Nguyen Van Ba, Chu Van Men, Nguyen Tien Khi, Tran Viet Tiet, Pham Gia
Khanh, Le Bach Quang, Nguyen Van Chuyen, Vu Dinh Tien, Nguyen Xuan
Nhiem & Phan Van Kiem
To cite this article: Nguyen Van Ba, Chu Van Men, Nguyen Tien Khi, Tran Viet Tiet, Pham Gia
Khanh, Le Bach Quang, Nguyen Van Chuyen, Vu Dinh Tien, Nguyen Xuan Nhiem & Phan Van
Kiem (2019): Flavonol glycosides from Fissistigmaꢀmaclurei, Journal of Asian Natural Products
Research, DOI: 10.1080/10286020.2019.1671374
To link to this article: https://doi.org/10.1080/10286020.2019.1671374
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2019.1671374
Flavonol glycosides from Fissistigma maclurei
a
a
b
a
ꢀ
ꢀ
Nguyen Van Ba , Chu Van Men , Nguyen Tien Khi , Tran Viet Tiet ,
a
a
a
c
Pham Gia Khanh , Le Bach Quang , Nguyen Van Chuyen , Vu Dinh Tien ,
Nguyen Xuan Nhiem and Phan Van Kiem
d
d
a
b
c
Vietnam Military Medical University, Hanoi, Vietnam; Phenikaa University, Hanoi, Vietnam; Vietnam
d
National Cancer Hospital, Hanoi, Vietnam; Institute of Marine Biochemistry, Vietnam Academy of
Science and Technology, Hanoi, Vietnam
ABSTRACT
ARTICLE HISTORY
Two new flavonol glycosides, fissmacosides A (1) and B (2) along
with two known flavonol glycosides, kaempferol 3-O-a-L-rhamno-
pyranosyl-(1!6)-b-D-galactopyranoside (3) and kaempferol 3-O-
b-D-glucopyranosyl-(1!4)-a-L-rhamnopyranosyl-(1!6)-[4-(E)-feru-
loyl]-b-D-galactopyranoside (4) were isolated from the methanol
extract of the leaves of Fissistigma maclurei Merr. Their structures
were determined on the basis of extensive spectroscopic meth-
ods, including 1D-, 2D-NMR, and MS data.
Received 3 May 2019
Accepted 18 September 2019
KEYWORDS
Flavonol; Fissistigma
maclurei; fissmacoside A;
fissmacoside B
1. Introduction
Fissistigma is one of the important genera of Annonaceae family. There are about 80
Fissistigma species distributed in Australia and Asia, especially in South-East Asia
such as Thailand, Malaysia, Cambodia, and Vietnam. Chemical studies of Fissistigma
genus indicated the main components to be flavonoids [1], alkaloids [2], and sesqui-
terpenes [3]. These compounds exhibited antioxidant and anticancer activities.
Fissistigma maclurei Merr. are climbers that grow up to 6 m tall. There are few
reports of pharmacological potential and chemistry of this plant [4,5]. This article
reports the isolation and structure elucidation of two new and two known flavonol
glycosides from the leaves of F. maclurei Merr (Figure 1).
CONTACT Phan Van Kiem
phankiem@yahoo.com
Institute of Marine Biochemistry, Vietnam Academy of
Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
ꢀ
These authors contributed equally to this work.
Supplemental data for this article can be accessed at https://doi.org/10.1080/10286020.2019.1671374.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
N. V. BA ET AL.
Figure 1. Chemical structures of compounds 1 and 2.
2. Results and discussion
Compound 1 was obtained as a yellow amorphous powder and its molecular formula
was determined to be C H O from HR-ESI-MS ion peak at m/z 809.2110
3
4
42 21
þ
1
[
M þ Na] . The H-NMR spectrum of 1 (in methanol-d ) showed the specific signals:
4
two aromatic protons of A ring at d 6.19 (1 H, br s) and 6.41 (1 H, br s); three aro-
H
matic protons of ABX coupling system of B ring at d 6.89 (1 H, d, J ¼ 8.4 Hz), 7.59
H
(
1 H, d, J ¼ 8.4 Hz), and 8.02 (1 H, s); one methoxy group at d 3.94 (3 H, s); three
H
anomeric protons at dH 4.48 (1 H, d, J ¼ 7.6 Hz), 4.50 (1 H, br s), and 5.20 (d,
J ¼ 7.8 Hz) and one secondary methyl group at d 1.20 (3 H, d, J ¼ 6.0 Hz), suggesting
H
1
3
the presence of three sugar moieties. The C-NMR and HSQC spectra of 1 revealed
the signals of 34 carbons, of which 15 carbons were assigned to a flavonol and 18 car-
bons to three monosaccharide moieties (Table 1). The HMBC correlations between
0
0
0
0
H-2 (d 8.02)/H-6 (d 7.59) and C-2 (dC 158.9)/C-1 (d 122.9)/C-4 (d 150.9);
between H-5 (d 6.89) and C-3 (d 148.4); and between the methoxy (d 3.94) and
C-3 (d 148.4) suggested that the methoxy and the hydroxy were at C-3 and C-4 ,
H
H
C
C
0
0
H
C
H
0
0
0
C
respectively (Figure 2). Thus, the flavonol aglycone was identified as isorhamnetin
[
6]. Acid hydrolysis of 1 revealed D-glucose, D-galactose, and L-rhamnose (identified
as trimethylsilyl (TMS) derivatives by a gas chromatography method). In addition,
0
00
the multiplicity of gal H-1ʺ [5.20 (d, J ¼ 7.8 Hz], rha H-1 [4.50 (br s)], and glc H-1ʺ
1
ʺ [4.48 (d, J ¼ 7.6 Hz)] in the H-NMR spectrum of 1 proved configurations of sugar
moieties as b-D-galactopyranosyl, b-D-glucopyranosyl, and a-L-rhamnopyranosyl. The
0
00
00
0000
HMBC correlations between rha H-1 (dH 4.50) and gal C-6 (d 68.0); glc H-1
dH 4.48) and rha C-4 (dC 83.4); and between gal H-1ʺ (dH 5.20) and C-3 (dC
C
000
(
1
35.5) suggested the sugar linkages as O-b-D-glucopyranosyl-(1!4)-a-L-rhamnopyra-
nosyl-(1!6)-b-D-galactopyranosyl and at C-3 of flavonol [7]. Consequently, the new
compound 1 was determined to be isorhamnetin 3-O-b-D-glucopyranosyl-(1!4)-a-L-
rhamnopyranosyl-(1!6)-b-D-galactopyranoside and named as fissmacoside A.
The HR-ESI-MS experiment of 2 resulted to the molecular formula of C H O .
44
48 23
1
The H-NMR spectrum suggested that compound 2 was a flavonol glycoside with the
following signals: two meta-protons of A ring at d 6.20 (1 H, s) and 6.42 (1 H, s); an
H

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
1
13
Table 1. H- and C-NMR spectroscopic data for compounds 1 and 2 in methanol-d4.
1
2
C
d
C
d
H
(mult., J ¼ Hz)
d
C
d
H
(mult., J ¼ Hz)
Flavonol
2
3
4
5
6
7
8
9
158.9
135.5
179.5
163.0
100.0
166.0
94.9
158.5
105.7
122.9
114.6
148.4
150.9
116.0
123.8
57.0
–
–
–
–
159.0
135.4
179.3
163.0
100.0
166.0
95.0
158.5
105.7
123.0
114.9
148.3
151.1
116.0
123.9
57.0
–
–
–
–
6.19 (s)
6.41 (s)
6.20 (s)
6.42 (s)
–
–
–
–
–
–
–
–
1
1
2
3
4
5
6
3
3
1
2
3
4
5
6
0
0
0
0
0
0
0
0
8.02 (s)
8.05 (s)
–
–
–
–
6.89 (d, 8.4)
7.59 (d, 8.4)
3.94 (s)
6.91 (d, 8.4)
7.63 (d, 8.4)
3.96 (s)
-OMe
-OGal
0
0
0
0
0
0
0
0
0
0
0
0
104.9
73.0
75.0
70.1
75.5
68.0
5.20 (d, 7.8)
105.0
73.5
73.5
71.4
73.9
67.1
5.29 (d, 6.0)
3.81–3.85 (m)
3.83–3.85 (m)
5.38 (br s)
3.88–3.90 (m)
3.21–3.25 (m)
3.47–3.50 (m)
3.78–3.82 (m)
3.53–3.55 (m)
3.75 (br d, 2.8)
3.63–3.66 (m)
3.47–3.49 (m)
3
.69–3.71 (m)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6
1
2
3
4
5
6
4
1
2
3
4
5
6
-ORha
00
00
00
00
00
00
00
000
000
000
000
000
000
102.0
72.0
72.3
83.4
68.4
18.2
4.50 (br s)
3.58 (br s)
3.67 (dd, 2.0, 8.0)
3.45–3.49 (m)
3.55–3.59 (m)
1.20 (d, 6.0)
102.3
72.0
72.0
82.8
68.2
18.2
4.46 (br s)
3.62 (br s)
3.70 (dd, 2.0, 8.0)
3.42–3.46 (m)
3.41–3.45 (m)
1.09 (d, 5.0)
-OGlc
105.7
76.0
78.3
71.6
78.0
62.8
4.48 (d, 7.6)
105.2
75.9
78.2
71.7
77.9
62.9
4.54 (d, 7.6)
3.14–3.18 (m)
3.30–3.32 (m)
3.24–3.28 (m)
3.19–3.23 (m)
3.62 (dd, 4.8, 12.0)
3.19–3.23 (m)
3.33–3.37 (m)
3.22–3.26 (m)
3.20–3.24 (m)
3.59 (dd, 4.0, 12.0)
3.79 (br d, 12.0)
3
.81 (br d, 12.0)
0
0
0
0
0
0
0
0
0
0
0
4
1
2
3
4
5
6
7
8
9
-OFer
0000
0000
0000
0000
0000
0000
0000
0000
0000
127.7
111.6
149.4
150.8
116.5
124.5
147.8
115.1
168.8
56.3
–
7.20 (s)
–
–
6.83 (d, 8.0)
7.10 (d, 8.0)
7.64 (d, 16.0)
6.38 (d, 16.0)
–
OMe
3.93 (s)
Assignments were done by HSQC, HMBC, and COSY experiments; Glc, glucopyranosyl; Rha, rhamnopyranosyl; Gal,
galactopyranosyl; Fer, feruloyl.
ABX coupling system of B ring at dH 6.91 (1 H, d, J ¼ 8.4 Hz), 7.63 (1 H, d,
J ¼ 8.4 Hz), and 8.05 (1 H, s); one methoxy group at d 3.96 (3 H, s); three anomeric
H
protons at d 4.46 (1 H, br s), 4.54 (1 H, d, J ¼ 7.6 Hz), and 5.29 (1 H, d, J ¼ 6.0 Hz);
H
the proton signals of (E)-feruloyl moiety at d 7.20 (1 H, s), 6.83 (1 H, d, J ¼ 8.0 Hz),
H

4
N. V. BA ET AL.
Figure 2. The key HMBC and COSY correlations of compounds 1 and 2.
7
.10 (1 H, d, J ¼ 8.0 Hz), 6.38 (1 H, d, J ¼ 16.0 Hz), and 7.64 (1 H, d, J ¼ 16.0 Hz), and
13
one methoxy group at d 3.93 (s). The C-NMR and HSQC spectra of 2 showed the
H
signals of 44 carbons, including 2 carbonyl, 12 non-protonated carbons, 25 methines, 2
1
13
methylenes, and 3 methyl carbons (Table 1). The H- and C-NMR data of 2 showed
almost the same to those of fissmacoside A (1) except for the addition of feruloyl moiety
at gal C-4ddition of feruloyl(arb2 proved the presence of D-galactose, L-rhamnose, and
D-glucose (identified as TMS derivatives by a gas chromatography method). The HMBC
0
00
00
0000
correlations between rha H-1 (dH 4.46) and gal C-6 (d 67.1); glc H-1 (dH 4.54)
C
0
00
00
and rha C-4 (d 82.8); and between gal H-4 (d 5.38) and fer C-98) and d 168.8)
C
H
C
confirmed the sugar linkages as O-b-D-glucopyranosyl-(1!4)-a-L-rhamnopyranosyl-
1!6)-b-D-galactopyranosyl and feruloyl group at gal C-4ʺ. Finally, the HMBC correl-
ation from gal H-1ʺ (d 5.29) to C-3 (d 135.4) indicated the trisaccharide position at
(
H
C
C-3 of flavonol. Based on the above evidence, compound 2 was determined to be iso-
rhamnetin 3-O-b-D-glucopyranosyl-(1!4)-a-L-rhamnopyranosyl-(1!6)-[4-(E)-feruloyl]-
b-D-galactopyranoside, a new compound and named as fissmacoside B.
The known compounds were elucidated as kaempferol 3-O-a-L-rhamnopyranosyl-
(
1!6)-b-D-galactopyranoside (3) [8] and kaempferol 3-O-b-D-glucopyranosyl-(1!4)-
a-L-rhamnopyranosyl-(1!6)-[4-(E)-feruloyl]-b-D-galactopyranoside (4) [7] by compar-
ing their observed and reported physical data.
All isolated compounds were evaluated for cytotoxic activity against three human
cancer cell lines, A-549 (human lung cancer), MCF-7 (human breast cancer), and
MKN-45 (human gastric cancer) at 10 mM. However, none of them showed cytotoxic
activity (cell viability >50%).
3. Experimental
3.1. General experimental procedures
Optical rotations were determined on a JASCO P-2000 digital polarimeter (JASCO Ltd.,
Tokyo, Japan). UV spectra were obtained from Labtron LUS-B14 spectrophotometer
(Labtron Equipment Ltd., Camberley, UK). The IR spectra were obtained from a Bruker
TENSOR 37 FT-IR spectrometer (Bruker, Ettlingen, Germany). NMR spectra were

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
recorded on an Agilent 400 MHz (Agilent Technologies, Santa Clara, CA, USA). Data
processing was carried out with the MestReNova ver. 9.0.1. HR-ESI-MS were recorded
on an AGILENT 6550 iFunnel Q-TOF LC/MS system (Agilent Technologies). HPLC
was carried out using an AGILENT 1200 HPLC system (Agilent Technologies). Column
chromatography was performed on silica gel (Kieselgel 60, 230–400 mesh, Merck,
Darmstadt, Germany) or RP-18 resins (30–50lm, Fuji Silysia Chemical Ltd., Tokyo,
Japan). For thin layer chromatography (TLC), pre-coated silica-gel 60 F254 (0.25 mm,
Merck) and RP-18 F254S (0.25 mm, Merck) plates were used.
3
.2. Plant material
The leaves of Fissistigma maclurei Merr. were collected at Curut, Daknong, Vietnam
in April 2018, and identified by Dr. Nguyen, The Cuong, Institute of Ecology and
Biological Resources, VAST. A voucher specimen (HVQY1804) was deposited at the
Military Medical University.
3
.3. Extraction and isolation
The dried powdered leaves of F. maclurei (2.0 kg) were sonicated with hot methanol
two times (each 5 L, 2 h) and removed the solvent in vacuo to yield methanol
(
MeOH) extract (120.0 g). The MeOH extract was suspended in H O (1.0 L) and suc-
2
cessively partitioned with chloroform, and ethyl acetate (EtOAc) to yield chloroform
(FM1, 18.0 g), EtOAc (FM2, 8.0 g) residues, and water layer (FM3).
The water-soluble fraction (FM3) was chromatographed on a Diaion HP-20P column
eluting with water then increasing the concentration of MeOH in water (25, 50, 75, and
00%) to obtain four sub-fractions, FM3A (5.0 g), FM3B (8.0 g), FM3C (16.0 g), and
1
FM3D (7.0 g). The FM3C fraction was chromatographed on a silica gel column eluting
with gradient solvents of CHCl –MeOH (10: 1 M3C fraction was chromatographed on
3
a silica, FM3C1–FM3C3. The FM3C2 fraction was chromatographed on a Sephadex
LH-20 column eluting with MeOH to give two fractions, FM3C2A-FM3C2B. The frac-
tion FM3C2A was chromatographed on HPLC using J’sphere ODS-H80 (150 mm ꢁ
20mm ID) with solvent condition of 35% acetonitrile in water, the flow rate of 3 ml/
min, and the wavelength of 254 nm to yield compound 2 (5.0 mg, t 38.8 min). The
R
FM3C3 fraction was chromatographed on a RP-18 column using MeOH–water (1: 1, v/
v) as eluent solvent to give three fractions, FM3C3A-FM3C3C. The FM3C3B was chro-
matographed on HPLC using J’sphere ODS-H80 (150 mm ꢁ 20 mm ID) with solvent
condition of 25% acetonitrile in water, the flow rate of 3 ml/min, and the wavelength of
254 nm to yield compounds 1 (5.0 mg, tR 44.2 min) and 3 (11.0 mg, tR 51.7 min).
Compound 4 (5.0 mg, t 32.3 min) was obtained from FM3C3C using J’sphere ODS-
R
H80 column (150 mm ꢁ 20 mm ID) on Agilent HPLC 1290 with solvent condition of
30% acetonitrile in water, the flow rate of 3 ml/min, and the wavelength of 254 nm
3
.3.1. Fissmacoside A (1)
2
5
Yellow amorphous powder. [a]D ꢂ67.0 (c 0.1 MeOH). UV (MeOH) kmax: 264,
ꢂ1
1
13
370 nm. IR (KBr) ꢀmax: 3390, 1658 cm . H- and C-NMR spectral data (methanol-

6
N. V. BA ET AL.
d4): see Table 1. HR-ESI-MS m/z: 809.2110 [M þ Na]þ (calcd for
C H NaO , 809.2111).
3
4
42
21
3
.3.2. Fissmacoside B (2)
2
5
Yellow amorphous powder. [a]D ꢂ86.0 (c 0.1 MeOH). UV (MeOH) kmax: 265,
ꢂ1
1
13
370 nm. IR (KBr) ꢀmax: 3410, 1675, 1658 cm . H- and C-NMR spectral data
þ
(
methanol-d ): see Table 1. HR-ESI-MS m/z: 985.2606 [M þ Na] (calcd for
4
C H NaO , 985.2584).
4
4
50
24
3.4. Acid hydrolysis
Each compound (1 and 2, 2.0 mg) was separately dissolved in 1.0 N HCl
ꢃ
(
dioxane–H O, 1:1, v/v, 1.0 ml) and heated to 80 C in a water bath for 3 h. The
2
acidic solution was neutralized with silver carbonate; the solvent was thoroughly
removed under a nitrogen stream overnight. After extraction with CHCl , the water
3
layer was concentrated to dryness using N . The residue was dissolved in pyridine
2
(
0
0.1 ml) and then added L-cysteine methyl ester hydrochloride in pyridine (0.06 M,
ꢃ
.1 ml). The reaction mixture was heated at 60 C for 2 h and then added trimethylsi-
ꢃ
lylimidazole solution (0.1 ml), heating at 60 C for 1.5 h. The dried product was parti-
tioned with n-hexane and H O (0.1 ml each), and the organic layer was analyzed by
2
gas chromatography: column DB-5 (0.32 mm ID ꢁ 30 m length), detector FID, col-
ꢃ
ꢃ
ꢃ
umn temperature 210 C, injector temperature 270 C, detector temperature 300 C,
carrier gas He (2 ml/min). Under these conditions, the standard sugars gave peaks at
tR (min) 14.11 and 14.26 for D- and L-glucose, 18.71 and 22.92 for D- and L-galactose,
and 4.50 for L-rhamnose, respectively. Peaks at t (min) 14.11, 18.71, and 4.50 of
R
D-glucose, D-galactose, and L-rhamnose for all compounds were observed.
3
.5. Cytotoxic assays
See Reference [9].
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This article’s data dues to the state level research “Research on the intervention increasing the
ability to protect the public health in the national frontier in Tay Nguyen and to create goods
from the local herbs”. Code: TN16/T03.
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