Phytochemistry Letters 20 (2017) 119–122

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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 MARK
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

A R T I C L E I N F O A B S T R A C T

Keywords: From the leaves of Bidens pilosa L., the n-hexane, chloroform, and aqueous extracts exhibited in vitro α-
Bidens pilosa L. glucosidase inhibitory activity, with IC50 values of 235.8, 125.6, and 100.3 μg/mL, respectively. Two new
Asteraceae compounds, methyl 4-O-caffeoyl-2-C-methyl-D-erythronate (1) and 4-O-methylokanin (2), and seven known
Caffeoylquinic acid compounds were isolated from these extracts. The chemical structures of 1–9 were elucidated on the basis of
α-Glucosidase inhibitor
NMR spectroscopic analysis. The caffeoylquinic acid derivatives were isolated from the aqueous extract, and
showed significant α-glucosidase inhibitory activity with IC50 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 (1 and 2) and seven known compounds (3–9).
Compound 1 was obtained as a yellowish liquid with an optical
D −14 (c 0.02, MeOH). The HR-ESI-MS spectrum,
activity of [α ]25
acquired in the positive mode, showed a sodiated molecular ion peak
at m/z 349.0878 [M+Na]+ (calcd for C15H18O8Na, 349.0899). The 1H
NMR spectrum showed an ABX aromatic system [δH 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 [δH 6.20 (1H, d, J = 15.9 Hz,
H-8′), and 7.47 (1H, d, J = 15.9 Hz, H-7′)], one methylene group [δH
4.05 (1H, dd, J = 11.1, 7.6 Hz, H-4a) and δH 4.13 (1H, dd, J = 11.1,
4.2 Hz, H-4b)], one oxygenated methine proton [δH 3.89 (1H, m, H-3)],
one methoxy group [δH 3.63 (3H, s, H-6)], and one methyl group [δH
1.28 (3H, s, H-5)] (Table 1). The 13C NMR and HSQC spectra displayed
the signals of two carbonyl carbons [δC 174.9 (C-1) and 166.5 (C-9′)],
two oxygenated aromatic carbons [δC 148.5 (C-4′) and 145.2 (C-3′)],
one aromatic quaternary carbon [δC 125.5 (C-1′)], three aromatic
methine carbons [δC 121.4 (C-6′), 115.9 (C-5′), and 114.7 (C-2′)], two

⁎
Corresponding author.
E-mail address: ttquang@hcmus.edu.vn (Q.T. That).

http://dx.doi.org/10.1016/j.phytol.2017.04.015
Received 9 February 2017; Accepted 13 April 2017
Available online 04 May 2017
1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
T. Van Nguyen Thien et al. Phytochemistry Letters 20 (2017) 119–122

Table 1 Table 2
1 13 1
H (500 MHz) and C (125 MHz) NMR spectroscopic data of compound 1 in DMSO-d6. H (500 MHz) and 13C (125 MHz) NMR spectroscopic data of compound 2 and okanin-4-
methoxy-4′-O-glucopyranoside in DMSO-d6.
Position 1
Position 2 Okanin-4-methoxy-4′-O-
δC δH (J in Hz) glucopyranoside

1 174.9 δH, (J in Hz) δC δH, J (Hz) δC

2 75.6
3 72.4 3.89 (1H, m) 1 127.6 127.5
4 64.7 4.13 (1H, dd, 11.1, 4.3) 2 7.33 (1H, d, 2.1) 115.1 7.36 (1H, s) 115.3
4.05 (1H, dd, 11.1, 7.6) 3 146.6 146.7
5 21.5 1.28 (3H, s) 4 150.3 150.7
6 51.9 3.63 (3H, s) 5 6.99 (1H, d, 8.4) 111.9 7.01 (1H, d, 8.0) 111.9
1′ 125.5 6 7.30 (1H, dd, 8.4, 122.1 7.34 (1H, d, 8.0) 122.5
2′ 114.7 7.03 (1H, d, 1.9) 2.1)
3′ 145.2 4.60 (1H, t, 2.5) 1′ 113.4 115.7
4′ 148.5 2′ 153.5 152.5
5′ 115.9 6.77 (1H, d, 8.2) 3′ 132.4 143.3
6′ 121.4 6.98 (1H, dd, 8.2, 1.9) 4′ 152.6 150.6
7′ 145.7 7.47 (1H, d, 15.9) 5′ 6.42 (1H, d, 8.9) 107.6 6.78 (1H, d, 9.5) 106.5
8′ 113.9 6.20 (1H, d, 15.9) 6′ 7.70 (1H, d, 8.9) 122.4 7.81(1H, d, 9.5) 121.7
9′ 166.5 C]O 192.0 192.8
α 7.65–7.75 (1H, m) 118.5 7.71 (1H, d, 15.8) 118.6
β 7.65–7.75 (1H, m) 144.0 7.79 (1H, d, 15.8) 144.9
olefinic carbons [δC 145.7 (C-7′) and 113.9 (C-8′)], four oxygenated 2′-OH 13.53 (1H, s)
4-OCH3 3.84 (3H, s) 55.7 3.85 (3H, s) 55.7
carbons [δC 75.6 (C-2), 72.4 (C-3), 64.7 (C-4), and 51.9 (C-6), and one
1″ 4.92 (1H, d, 7.5) 101.0
methyl [δC 21.5 (C-5ic quaternary carbon [δC 125.5 (C-1′)], three 2″ 3.35–3.20 (1H, m) 73.2
aromatic methine carbons [δC 121.4 (C-6′), 115.9 (C-5′), and 114.7 (C- 3″ 3.35–3.20 (1H, m) 75.9
2′)], two olefinic carbons [δC 145.7 (C-7′) and 113.9 (C-8′)], four 4″ 3.35–3.20 (1H, m) 69.8
5″ 3.35–3.20 (1H, m) 77.4
oxygenated carbons [δC 75.6 (C-2), 72.4 (C-3), 64.7 (C-4), and 51.9 (C-
6″ 3.49 (1H, m) 60.7
6), and one methyl [δC 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 Compound 4–9 showed the α-glucosidase inhibitory activity with
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, IC50 values ranging from 10.7 to 208.5 μM. The 4-O-caffeoyl group of D-
and H-6/C-1 showed the opening of 2,3-dihydroxy-2-methyl-γ-butyr- erythronic acid derivatives were found to decrease the activity
olactone ring (Gogoi and Argade, 2006), and deduced the locations of (5 > 1); moreover, the presence of γ-lactone has a slightly increased
the caffeoyl and the methoxy groups at C-4 and C-1. Moreover, the effect on activity. The caffeoylquinic acid derivatives showed more
NOESY experiment (Supporting information) was recorded but it could potent inhibitory activity than that of acarbose (IC50, 214.5 μM). The
not conclude the relative configuration of 1. Based on the reported presence of caffeoyloxy groups at both C-4 and C-5 of quinic acid
structures of the caffeoyl derivatives from B. pilosa (Ogawa and Sashida, derivatives significantly increases the activity (7 > > 6 > 8 > 9)
1992), the structure of 1 was suggested as methyl 4-O-caffeoyl-2-C- (Table 3).
methyl-D-erythronate. In this research, we reported the structures of two new compounds,
Compound 2 was obtained as a yellowish powder. It was deduced to methyl 4-O-caffeoyl-2-C-methyl-D-erythronate (1) and 4-O-methyloka-
have molecular formula C16H14O6 based on the HR-ESI–MS spectrum at nin (2), together with seven known compounds (3–9). All isolated
m/z 325.0696 [M+Na]+ (calcd for C16H14O6Na, 325.0688). The 1H compounds were evaluated the α-glucosidase inhibitory activity.
NMR spectrum showed the signals of an ABX aromatic system [δH 6.99 Compound 7, a dicaffeoylquinic acid derivative, showed the most
(1H, d, J = 8.4 Hz, H-5), 7.30 (1H, dd, J = 8.4, 2.1 Hz, H-6), and 7.33 potent activity with IC50 values of 10.7 μM, comparable to those of a
(1H, d, J = 2.1 Hz, H-2)], two olefinic protons [δH 7.65–7.75 (2H, m, positive control acarbose (IC50, 214.5 μM).
Hα, Hβ)], two aromatic methines [δH 6.42 (1H, d, J = 8.9 Hz, H-5′),
7.70 (1H, d, J = 8.9 Hz, H-6′)], a hydroxy group at δH 13.53, and a 3. Experimental
methoxy group at δH 3.84. The 13C NMR and HSQC spectra showed the
resonances of five oxygenated aromatic carbons [δC 153.5 (C-2′), 152.6 3.1. General experimental procedures
(C-4′), 150.3 (C-4), 146.6 (C-3), and 132.4 (C-3′)], two quaternary
aromatic carbons [δC 113.4 (C-1′) and 127.6 (C-1)], five aromatic The NMR spectra were acquired on a Bruker Avance III 500 MHz
methine carbons [δC 107.6 (C-5′), 122.4 (C-6′), 115.1 (C-2), 111.9 (C- spectrometer with tetramethylsilane (TMS) as an internal standard,
5), and 122.1 (C-6)], one methoxy carbon [δC 55.7 (4-OCH3)], two with chemical shifts expressed in δ (ppm) values. The HR-ESI–MS were
olefinic carbons [δC 118.5 (Cα) and 144.0 (Cβ)], and one carbonyl determined with a MicrOTOF QII mass spectrometer (Bruker Daltonics).
carbon (δC 192.0), suggesting the presence of a chalconoid skeleton The optical rotation values were measured on a Kruss digital polari-
(Table 2). The 1H and 13C NMR data of 2 resembled closely those of meter. Analytical and preparative TLC were performed on precoated
okanin (Kim et al., 2014; Pardede et al., 2016) except for the presence Merck Kieselgel 60 F254 or RP-18 F254 plates (0.25 mm or 0.5 mm
of a methoxy group. The A-ring was suggested as a tetrasubstituted ring thickness).
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′, 3.2. Chemicals
and H-5′/C-3′ (Fig. 2). The HMBC correlations between H-6/C-2, H-6/
C-β, H-6/C-4 and OCH3/C-4 indicated the position of a methoxy group α-Glucosidase (EC 3.2.1.20) from Saccharomyces cerevisiae (750
at C-4 of the B-ring. It was confirmed by comparison the NMR data of 2 UN), and p-nitrophenyl-α-D-glucopyranoside were obtained from
with those of the aglycone of okanin-4-methoxy-4′-O-glucopyranoside Sigma Chemical Co. (St. Louis, MO, USA). Acarbose and DMSO were
(Redl et al., 1992). Thus, compound 2 assigned as 4-O-methylokanin purchased from Merck (Darmstadt, Germany). Other chemicals were of
(Fig. 1). the highest grade available.

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T. Van Nguyen Thien et al. Phytochemistry Letters 20 (2017) 119–122

Fig 1. Structures of compound 1–9.

3.3. Plant material Table 3

α-Glucosidase inhibitory activities of the isolated compounds.
The leaves of Bidens pilosa L. were collected in Ho Chi Minh City,
Compound IC50 (μM)
Vietnam in November 2011. The plant was identified by late pharma-
cist and botanist Binh Duc Phan. A voucher specimen (BP001) was Methyl 4-O-caffeoyl-2-C-methyl-D-erythronate (1) > 250
deposited in the herbarium of the Department of Organic Chemistry, 4-Methoxyokanin (2) > 250
VNUHCM–University of Science. Centaureidin (3) > 250
Jaceidin (4) 208.5
3-O-Caffeoyl-2-C-methyl-D-erythrono-1,4-lactone (5) 149.8
3.4. Extraction and isolation 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
Dried leaf powder of Bidens pilosa L. (8 kg) was exhaustively Methyl 5-O-E-caffeoylquinate (9) 74.7
extracted with methanol. The methanol residue (1.2 kg; IC50, Acarbosea 214.5
220 μg mL−1) was suspended in H2O and successively partitioned with
a
n-hexane and chloroform, to yield the n-hexane (300 g; IC50, Positive control.
235.8 μg mL−1), chloroform (130 g; IC50, 125.6 μg mL−1), and aqueous
(400 g; IC50, 100.3 μg mL−1) extracts, respectively. The chloroform to give seven fractions (AQ2.1–2.7). Subfraction AQ2.6 (1.4 g) was
extract was subjected over a silica gel column eluted with CHCl3/MeOH separated over a sephadex LH-20 column with MeOH, and further
mixtures (9.5:0.5 to 0:10, v/v), to afford five fractions (C1–5). Fraction purified by preparative RP-TLC with CH3CN–H2O (65:35, v/v), to
C2 (20.2 g) was chromatographed over a silica gel column with CHCl3/ afford compounds 6 (5 mg), 7 (15 mg), 8 (5 mg), and 9 (14 mg).
MeOH mixtures (8:2 to 0:10, v/v), to obtain eight subfractions Methyl 4-O-caffeoyl-2-C-methyl-D-erythronate (1): yellowish liquid,
D – 14 (c 0.02, MeOH); H and
1 13
(C1.1–1.8). Subfraction C1.3 was subjected over a ODS column eluted [α ]25 C NMR (DMSO-d6, 500 MHz), see in
with MeOH–H2O (6:4, v/v) to give compounds 2 (6 mg), 3 (8 mg), and Table 1; HR-ESI–MS m/z 349.0878 [M+Na]+ (calcd for C15H18O8Na,
4 (5 mg). Subfraction C1.4 was purified by preparative TLC with n- 349.0899).
hexane–CHCl3–EtOAc (3:2:5), to yield compounds 1 (5 mg), and 5 4-O-Methylokanin (2): yellowish powder; 1H and 13C NMR (DMSO-
(12 mg). The aqueous extract was subjected over a silica gel column d6, 500 MHz), see in Table 2; HR-ESI–MS at m/z 325.0696 [M+Na]+
eluted with CHCl3/MeOH mixtures (9:1 to 0:10, v/v), to give seven (calcd for C16H14O6Na, 325.0688).
fractions (AQ1–7). Fraction AQ2 (110.7 g) was chromatographed over a
silica gel column eluted with CHCl3/MeOH mixtures (8:2 to 0:10, v/v),

Fig. 2. Significant HMBC correlations (solid arrows) observed for 1 and 2.

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T. Van Nguyen Thien et al.
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 Na2CO3 (375 μL). Enzymatic
activity was quantified by measuring absorbance at 401 nm. The IC50
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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