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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Two new compounds and α-glucosidase inhibitors from the leaves of Bidens
pilosa L.
Truong Van Nguyen Thien, Vi Ha Thi Huynh, Loan Kieu Thi Vo, Nhan Trong Tran,
Thuat My Luong, Tho Huu Le, Toan Phan Duc, Quang Ton That
⁎
Faculty of Chemistry, VNUHCM—University of Science, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City, Viet Nam
ARTICLE INFO
Keywords:
Bidens pilosa L.
Asteraceae
Caffeoylquinic acid
α-Glucosidase inhibitor
ABSTRACT
From the leaves of Bidens pilosa L., the n-hexane, chloroform, and aqueous extracts exhibited in vitro α-
glucosidase inhibitory activity, with IC
50
values of 235.8, 125.6, and 100.3 μg/mL, respectively. Two new
compounds, methyl 4-O-caffeoyl-2-C-methyl-D-erythronate (1) and 4-O-methylokanin (2), and seven known
compounds were isolated from these extracts. The chemical structures of 1–9were elucidated on the basis of
NMR spectroscopic analysis. The caffeoylquinic acid derivatives were isolated from the aqueous extract, and
showed significant α-glucosidase inhibitory activity with IC
50
values ranging from 10.7 to 74.7 μM.
1. Introduction
Bidens pilosa L., belonging to Asteraceae is a perennial herb and an
esculent plant (Bartolome et al., 2013) and grows wild in Vietnam. All
parts of this herb have been used as the traditional medicine for
inflammation, immunological disorders, digestive disorders, infectious
diseases, cancers, metabolic syndrome, and wounds (Bartolome et al.,
2013). Previously, many polyacetylenes, flavonoids, caffeoylquinic and
p-coumaric acid derivatives, sesquiterpenes, pheophytins have been
reported (Xuan and Khanh, 2016). Moreover, B. pilosa was used as the
treatment of type I and type II diabetes mellitus (Connelly, 2009). The
aqueous extract of this herb was evaluated the activity on type II
diabetes (Hsu et al., 2009; Ubillas et al., 2000). In the process of
screening on Vietnamese medicinal plants for treatment of diabetes
mellitus (Dang et al., 2014, 2015), the α-glucosidase inhibitory activity
has been evaluated to find the active extracts and compounds (Van de
Laar et al., 2005).
Herein, a bioactivity-guided fractionation was carried out, leading
to the isolation of two new compounds, methyl 4-O-caffeoyl-2-C-
methyl-D-erythronate (1) and 4-O-methylokanin (2). The structures of
seven known compounds (3–9) were identified as centaureidin (3)
(Barberá et al., 1986), jaceidin (4)(Flamini et al., 2001), 3-O-caffeoyl-
2-C-methyl-D-erythrono-1,4-lactone (5)(
Ogawa and Sashida, 1992),
methyl 3,4-di-O-caffeoylquinate (6)(
Liu et al., 2013), methyl 4,5-di-O-
caffeoylquinate (7)(
Chen et al., 2014), methyl-3,5-di-O-caffeoylquinate
(8)(
Liu et al., 2013), methyl 5-O-E-caffeoylquinate (9)(Lee et al.,
2013). The methanol residue, all extracts, and isolated compounds were
evaluated the α-glucosidase inhibitory activity. This is the first report
about the α-glucosidase inhibitory activity of the leaves extract from
Bidens pilosa L.
2. Results and discussion
The dried powder of the leaves of Bidens pilosa was exhaustively
extracted with methanol. The methanol residue was fractionated into
the n-hexane, chloroform, and aqueous extracts. Further separation and
purification of the chloroform and aqueous extracts led to the isolation
of two new compounds (1and 2) and seven known compounds (3–9).
Compound 1was obtained as a yellowish liquid with an optical
activity of
α
[
]
D
2
5
−14 (c0.02, MeOH). The HR-ESI-MS spectrum,
acquired in the positive mode, showed a sodiated molecular ion peak
at m/z349.0878 [M+Na]
+
(calcd for C
15
H
18
O
8
Na, 349.0899). The
1
H
NMR spectrum showed an ABX aromatic system [δ
H
6.77 (1H, d,
J= 8.2 Hz, H-5′), 6.98 (1H, dd, J= 8.2, 2.0 Hz, H-6′), and 7.03 (1H, d,
J= 1.9 Hz, H-2′)], two olefinic protons [δ
H
6.20 (1H, d, J= 15.9 Hz,
H-8′), and 7.47 (1H, d, J= 15.9 Hz, H-7′)], one methylene group [δ
H
4.05 (1H, dd, J= 11.1, 7.6 Hz, H-4a) and δ
H
4.13 (1H, dd, J= 11.1,
4.2 Hz, H-4b)], one oxygenated methine proton [δ
H
3.89 (1H, m, H-3)],
one methoxy group [δ
H
3.63 (3H, s, H-6)], and one methyl group [δ
H
1.28 (3H, s, H-5)] (Table 1). The
13
C NMR and HSQC spectra displayed
the signals of two carbonyl carbons [δ
C
174.9 (C-1) and 166.5 (C-9′)],
two oxygenated aromatic carbons [δ
C
148.5 (C-4′) and 145.2 (C-3′)],
one aromatic quaternary carbon [δ
C
125.5 (C-1′)], three aromatic
methine carbons [δ
C
121.4 (C-6′), 115.9 (C-5′), and 114.7 (C-2′)], two
http://dx.doi.org/10.1016/j.phytol.2017.04.015
Received 9 February 2017; Accepted 13 April 2017
⁎
Corresponding author.
E-mail address: ttquang@hcmus.edu.vn (Q.T. That).
Phytochemistry Letters 20 (2017) 119–122
1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
MARK
olefinic carbons [δ
C
145.7 (C-7′) and 113.9 (C-8′)], four oxygenated
carbons [δ
C
75.6 (C-2), 72.4 (C-3), 64.7 (C-4), and 51.9 (C-6), and one
methyl [δ
C
21.5 (C-5ic quaternary carbon [δ
C
125.5 (C-1′)], three
aromatic methine carbons [δ
C
121.4 (C-6′), 115.9 (C-5′), and 114.7 (C-
2′)], two olefinic carbons [δ
C
145.7 (C-7′) and 113.9 (C-8′)], four
oxygenated carbons [δ
C
75.6 (C-2), 72.4 (C-3), 64.7 (C-4), and 51.9 (C-
6), and one methyl [δ
C
21.5 (C-5)]. The HMBC correlations between H-
7′/C-6′, H-7′/C-9′, H-8′/C-1′, and H-8′/C-9′were observed (Fig. 2),
which indicated the presence of a caffeoyl group. In addition, the HMBC
cross-peaks of H-4/C-3, H-4/C-9′, H-3/C-4, H-5/C-2, H-5/C-3, H-5/C-1,
and H-6/C-1 showed the opening of 2,3-dihydroxy-2-methyl-γ-butyr-
olactone ring (Gogoi and Argade, 2006), and deduced the locations of
the caffeoyl and the methoxy groups at C-4 and C-1. Moreover, the
NOESY experiment (Supporting information) was recorded but it could
not conclude the relative configuration of 1. Based on the reported
structures of the caffeoyl derivatives from B. pilosa (Ogawa and Sashida,
1992), the structure of 1was suggested as methyl 4-O-caffeoyl-2-C-
methyl-D-erythronate.
Compound 2was obtained as a yellowish powder. It was deduced to
have molecular formula C
16
H
14
O
6
based on the HR-ESI–MS spectrum at
m/z325.0696 [M+Na]
+
(calcd for C
16
H
14
O
6
Na, 325.0688). The
1
H
NMR spectrum showed the signals of an ABX aromatic system [δ
H
6.99
(1H, d, J= 8.4 Hz, H-5), 7.30 (1H, dd, J= 8.4, 2.1 Hz, H-6), and 7.33
(1H, d, J= 2.1 Hz, H-2)], two olefinic protons [δ
H
7.65–7.75 (2H, m,
H
α
,H
β
)], two aromatic methines [δ
H
6.42 (1H, d, J= 8.9 Hz, H-5′),
7.70 (1H, d, J= 8.9 Hz, H-6′)], a hydroxy group at δ
H
13.53, and a
methoxy group at δ
H
3.84. The
13
C NMR and HSQC spectra showed the
resonances of five oxygenated aromatic carbons [δ
C
153.5 (C-2′), 152.6
(C-4′), 150.3 (C-4), 146.6 (C-3), and 132.4 (C-3′)], two quaternary
aromatic carbons [δ
C
113.4 (C-1′) and 127.6 (C-1)], five aromatic
methine carbons [δ
C
107.6 (C-5′), 122.4 (C-6′), 115.1 (C-2), 111.9 (C-
5), and 122.1 (C-6)], one methoxy carbon [δ
C
55.7 (4-OCH
3
)], two
olefinic carbons [δ
C
118.5 (C
α
) and 144.0 (C
β
)], and one carbonyl
carbon (δ
C
192.0), suggesting the presence of a chalconoid skeleton
(Table 2). The
1
H and
13
C NMR data of 2resembled closely those of
okanin (Kim et al., 2014; Pardede et al., 2016) except for the presence
of a methoxy group. The A-ring was suggested as a tetrasubstituted ring
with three hydroxy and one carbonyl groups based on the HMBC
correlations between 2′-OH/C-1′,2′-OH/C-2′,2′-OH/C-3′, H-5′/C-1′,
and H-5′/C-3′(Fig. 2). The HMBC correlations between H-6/C-2, H-6/
C-β, H-6/C-4 and OCH
3
/C-4 indicated the position of a methoxy group
at C-4 of the B-ring. It was confirmed by comparison the NMR data of 2
with those of the aglycone of okanin-4-methoxy-4′-O-glucopyranoside
(Redl et al., 1992). Thus, compound 2assigned as 4-O-methylokanin
(Fig. 1).
Compound 4–9showed the α-glucosidase inhibitory activity with
IC
50
values ranging from 10.7 to 208.5 μM. The 4-O-caffeoyl group of D-
erythronic acid derivatives were found to decrease the activity
(5>1); moreover, the presence of γ-lactone has a slightly increased
effect on activity. The caffeoylquinic acid derivatives showed more
potent inhibitory activity than that of acarbose (IC
50
, 214.5 μM). The
presence of caffeoyloxy groups at both C-4 and C-5 of quinic acid
derivatives significantly increases the activity (7>>6>8>9)
(Table 3).
In this research, we reported the structures of two new compounds,
methyl 4-O-caffeoyl-2-C-methyl-D-erythronate (1) and 4-O-methyloka-
nin (2), together with seven known compounds (3–9). All isolated
compounds were evaluated the α-glucosidase inhibitory activity.
Compound 7, a dicaffeoylquinic acid derivative, showed the most
potent activity with IC
50
values of 10.7 μM, comparable to those of a
positive control acarbose (IC
50
, 214.5 μM).
3. Experimental
3.1. General experimental procedures
The NMR spectra were acquired on a Bruker Avance III 500 MHz
spectrometer with tetramethylsilane (TMS) as an internal standard,
with chemical shifts expressed in δ(ppm) values. The HR-ESI–MS were
determined with a MicrOTOF QII mass spectrometer (Bruker Daltonics).
The optical rotation values were measured on a Kruss digital polari-
meter. Analytical and preparative TLC were performed on precoated
Merck Kieselgel 60 F
254
or RP-18 F
254
plates (0.25 mm or 0.5 mm
thickness).
3.2. Chemicals
α-Glucosidase (EC 3.2.1.20) from Saccharomyces cerevisiae (750
UN), and p-nitrophenyl-α-D-glucopyranoside were obtained from
Sigma Chemical Co. (St. Louis, MO, USA). Acarbose and DMSO were
purchased from Merck (Darmstadt, Germany). Other chemicals were of
the highest grade available.
Table 1
1
H (500 MHz) and
13
C (125 MHz) NMR spectroscopic data of compound 1in DMSO-d
6.
Position 1
δ
C
δ
H
(Jin Hz)
1 174.9
2 75.6
3 72.4 3.89 (1H, m)
4 64.7 4.13 (1H, dd, 11.1, 4.3)
4.05 (1H, dd, 11.1, 7.6)
5 21.5 1.28 (3H, s)
6 51.9 3.63 (3H, s)
1′125.5
2′114.7 7.03 (1H, d, 1.9)
3′145.2 4.60 (1H, t, 2.5)
4′148.5
5′115.9 6.77 (1H, d, 8.2)
6′121.4 6.98 (1H, dd, 8.2, 1.9)
7′145.7 7.47 (1H, d, 15.9)
8′113.9 6.20 (1H, d, 15.9)
9′166.5
Table 2
1
H (500 MHz) and
13
C (125 MHz) NMR spectroscopic data of compound 2and okanin-4-
methoxy-4′-O-glucopyranoside in DMSO-d
6.
Position 2Okanin-4-methoxy-4′-O-
glucopyranoside
δ
H
,(Jin Hz) δ
C
δ
H
,J(Hz) δ
C
1 127.6 127.5
2 7.33 (1H, d, 2.1) 115.1 7.36 (1H, s) 115.3
3 146.6 146.7
4 150.3 150.7
5 6.99 (1H, d, 8.4) 111.9 7.01 (1H, d, 8.0) 111.9
6 7.30 (1H, dd, 8.4,
2.1)
122.1 7.34 (1H, d, 8.0) 122.5
1′113.4 115.7
2′153.5 152.5
3′132.4 143.3
4′152.6 150.6
5′6.42 (1H, d, 8.9) 107.6 6.78 (1H, d, 9.5) 106.5
6′7.70 (1H, d, 8.9) 122.4 7.81(1H, d, 9.5) 121.7
C]O 192.0 192.8
α7.65–7.75 (1H, m) 118.5 7.71 (1H, d, 15.8) 118.6
β7.65–7.75 (1H, m) 144.0 7.79 (1H, d, 15.8) 144.9
2′-OH 13.53 (1H, s)
4-OCH
3
3.84 (3H, s) 55.7 3.85 (3H, s) 55.7
1″4.92 (1H, d, 7.5) 101.0
2″3.35–3.20 (1H, m) 73.2
3″3.35–3.20 (1H, m) 75.9
4″3.35–3.20 (1H, m) 69.8
5″3.35–3.20 (1H, m) 77.4
6″3.49 (1H, m) 60.7
T. Van Nguyen Thien et al. Phytochemistry Letters 20 (2017) 119–122
120
3.3. Plant material
The leaves of Bidens pilosa L. were collected in Ho Chi Minh City,
Vietnam in November 2011. The plant was identified by late pharma-
cist and botanist Binh Duc Phan. A voucher specimen (BP001) was
deposited in the herbarium of the Department of Organic Chemistry,
VNUHCM–University of Science.
3.4. Extraction and isolation
Dried leaf powder of Bidens pilosa L. (8 kg) was exhaustively
extracted with methanol. The methanol residue (1.2 kg; IC
50
,
220 μgmL
−1
) was suspended in H
2
O and successively partitioned with
n-hexane and chloroform, to yield the n-hexane (300 g; IC
50
,
235.8 μgmL
−1
), chloroform (130 g; IC
50
, 125.6 μgmL
−1
), and aqueous
(400 g; IC
50
, 100.3 μgmL
−1
) extracts, respectively. The chloroform
extract was subjected over a silica gel column eluted with CHCl
3
/MeOH
mixtures (9.5:0.5 to 0:10, v/v), to afford five fractions (C1–5). Fraction
C2 (20.2 g) was chromatographed over a silica gel column with CHCl
3
/
MeOH mixtures (8:2 to 0:10, v/v), to obtain eight subfractions
(C1.1–1.8). Subfraction C1.3 was subjected over a ODS column eluted
with MeOH–H
2
O (6:4, v/v) to give compounds 2(6 mg), 3(8 mg), and
4(5 mg). Subfraction C1.4 was purified by preparative TLC with n-
hexane–CHCl
3
–EtOAc (3:2:5), to yield compounds 1(5 mg), and 5
(12 mg). The aqueous extract was subjected over a silica gel column
eluted with CHCl
3
/MeOH mixtures (9:1 to 0:10, v/v), to give seven
fractions (AQ1–7). Fraction AQ2 (110.7 g) was chromatographed over a
silica gel column eluted with CHCl
3
/MeOH mixtures (8:2 to 0:10, v/v),
to give seven fractions (AQ2.1–2.7). Subfraction AQ2.6 (1.4 g) was
separated over a sephadex LH-20 column with MeOH, and further
purified by preparative RP-TLC with CH
3
CN–H
2
O (65:35, v/v), to
afford compounds 6(5 mg), 7(15 mg), 8(5 mg), and 9(14 mg).
Methyl 4-O-caffeoyl-2-C-methyl-D-erythronate (1): yellowish liquid,
α
[
]
D
2
5
–14 (c0.02, MeOH);
1
H and
13
C NMR (DMSO-d
6
, 500 MHz), see in
Table 1; HR-ESI–MS m/z349.0878 [M+Na]
+
(calcd for C
15
H
18
O
8
Na,
349.0899).
4-O-Methylokanin (2): yellowish powder;
1
H and
13
C NMR (DMSO-
d
6
, 500 MHz), see in Table 2; HR-ESI–MS at m/z325.0696 [M+Na]
+
(calcd for C
16
H
14
O
6
Na, 325.0688).
Fig 1. Structures of compound 1–9.
Fig. 2. Significant HMBC correlations (solid arrows) observed for 1and 2.
Table 3
α-Glucosidase inhibitory activities of the isolated compounds.
Compound IC
50
(μM)
Methyl 4-O-caffeoyl-2-C-methyl-D-erythronate (1) > 250
4-Methoxyokanin (2) > 250
Centaureidin (3) > 250
Jaceidin (4) 208.5
3-O-Caffeoyl-2-C-methyl-D-erythrono-1,4-lactone (5) 149.8
Methyl 3,4-di-O-caffeoylquinate (6) 52.1
Methyl 4,5-di-O-caffeoylquinate (7) 10.7
Methyl-3,5-di-O-caffeoylquinate (8) 70.8
Methyl 5-O-E-caffeoylquinate (9) 74.7
Acarbose
a
214.5
a
Positive control.
T. Van Nguyen Thien et al. Phytochemistry Letters 20 (2017) 119–122
121
3.5. α-Glucosidase inhibitory assay
The inhibitory activity of α-glucosidase was determined according
to the method of Dang et al. (Dang et al., 2015). 3 mM p-nitrophenyl-α-
D-glucopyranoside (25 μL) and 0.2 U/mL α-glucosidase (25 μL) in
0.01 M phosphate buffer (pH = 7) were added to the sample solution
(625 μL) to start the reaction. Each reaction was carried out at 37 °C for
30 min and stopped by adding 0.1 M Na
2
CO
3
(375 μL). Enzymatic
activity was quantified by measuring absorbance at 401 nm. The IC
50
values were defined as the concentration of α-glucosidase inhibitor that
inhibited 50% of α-glucosidase activity. Acarbose, a known α-glucosi-
dase inhibitor, was used as a positive control.
Acknowledgment
This research was supported by a grant from the Vietnam National
University Ho Chi Minh City (No. A2015-18-02) to M. T. T. N.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.phytol.2017.04.015.
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