New flavanol and cycloartane glucosides from Landoltia punctata

Article abstract of DOI:10.3390/molecules19056623

Chemical investigation on the constituents of Landoltia punctata led to the isolation and identification of 17 compounds, four of which were new and identified as (3β,24S)-9,19-cycloartane-3,22,24,25-Tetraol 3-O-[β-D-glucopyranosyl-(1→2)]-[β-Dglucopyranos

Full text of DOI:10.3390/molecules19056623

Molecules 2014, 19, 6623-6634; doi:10.3390/molecules19056623
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
New Flavanol and Cycloartane Glucosides from
Landoltia punctata
Nini Wang, Guobo Xu, Yang Fang, Tao Yang, Hai Zhao * and Guoyou Li *
Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology,
Chinese Academy of Sciences, Chengdu 610041, China
*
Authors to whom correspondence should be addressed; E-Mails: zhaohai@cib.ac.cn (H.Z.);
ligy@cib.ac.cn (G.L.); Tel./Fax: +86-28-82890829 (G.L.).
Received: 26 February 2014; in revised form: 14 May 2014 / Accepted: 16 May 2014 /
Published: 22 May 2014
Abstract: Chemical investigation on the constituents of Landoltia punctata led to the
isolation and identification of 17 compounds, four of which were new and identified as
(
3β,24S)-9,19-cycloartane-3,22,24,25-tetraol
3-O-[β-D-glucopyranosyl-(1→2)]-[β-D-
glucopyranosyl-(1→6)]-β-D-glucopyranoside (1), (3β,24S)-9,19-cycloartane-3,24,25-triol
3-O-[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→6)]-β-D-glucopyranoside (2),
3,4'-dihydroxy-7,3'-dimethoxyflavan-5-O-β-D-glucopyranoside (3) and 3,4'-dihydroxy-
4,7,3'-trimethoxyflavan-5-O-β-D-glucopyranoside (4). Their structures were elucidated by
spectroscopic, chemical, and biochemical methods. Thus, cycloartane triterpenoids were
discovered in the Lemnaceae family for the first time. Compound 3 showed antioxidant
capacity in the positively charged 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid
+•
radical (ABTS ) and superoxide anion radical scavenging assays.
Keywords: Landoltia punctata; flavanol glucoside; cycloartane glucoside; antioxidant
1
. Introduction
The plant of the Lemnaceae, commonly known as duckweed, is a kind of aquatic monocot
angiosperm and the smallest with flowers, which is widely distributed on the surface of still, slowly
flowing, and polluted waters around the World. In this family, there are 37 species, representing five
genera: Lemna, Landoltia, Spirodela, Wolffia and Wolffiella [1,2]. Duckweeds grow very fast and
proliferate quickly by budding, allowing them to colonize freshwater habitats rapidly and produce 13

Molecules 2014, 19
6624
to 38 metric tons/hectare/year dry weight of plant mass [3,4]. Since duckweeds can absorb pollutants
eg. N, P and heavy metals) from wastewater, they have been commonly used in the treatment of
(
domestic and animal wastewater streams for many years [5–7]. Meanwhile, because of the relatively
high starch and low lignin content in this plant, it has been proved to be an ideal bioresource for
bioethanol production [4,8]. Supported by the Minister of Science and Technology and the Major
Projects of Knowledge Innovation Program of Chinese Academy of Sciences, our group have been
focused on the exploitation of duckweed in the fields of biofuels and natural products.
In our previous study, Landoltia punctata demonstrated obvious advantages in the biofuel aspect
for its higher starch content and easier acquisition among the five genera in Lemnaceae [9]. Large
scale cultivation of L. punctata has been carried out by our group, and tons of L. punctata biomass can
be obtained in a very short time. It is no doubt that L. punctata has become a new resource for natural
products such as proteins, polysaccharides, amino acids, and other small molecules.
There are a few reports about the chemical composition of L. punctata. To date, only eight
compounds have been reported. One is an anthocyanin, and the other seven isolated by paper
chromatography were determined by R values, UV spectral maxima and color reactions to be
f
saponarin, isosaponarin, saponaretin and the glucosides of saponaretin and vitexin [10]. Detailed
chemical exploration on this plant is imperative. In this study, we investigated the chemical
components of L. punctata by chromatographic and spectroscopic methods. As a result, four new
compounds: (3β,24S)-9,19-cycloartane-3,22,24,25-tetraol 3-O-[β-D-glucopyranosyl-(1→2)]-[β-D-
glucopyranosyl-(1→6)]-β-D-glucopyranoside (1), (3β,24S)-9,19-cycloartane-3,24,25-triol 3-O-[β-D-
glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→6)]-β-D-glucopyranoside (2), 3,4'-dihydroxy-7,3'-
dimethoxyflavan-5-O-β-D-glucopyranoside (3) and 3,4'-dihydroxy-4,7,3'-trimethoxyflavan-5-O-β-D-
glucopyranoside (4), (Figure 1), together with 13 known ones apigenin (5), luteolin (6), apigenin-7-O-
β-glucoside (7), luteolin-7-O-β-glucoside (8) [11], vitexin (9), isovitexin (10), orientin (11), isoorientin
(
12) [12], 6,8-di-C-β-glucosylapigenin (13) [13], 6-C-β-glucosyl-8-C-β-galactosylapigenin (14) [14],
β-sitosterol (15), stigmasterol (16) [15], 5,22-diene-3,6-dicarbonyl-stigmasterol (17) [16] were isolated
and identified from the 95% ethanol extract of L. punctata.
Figure 1. Chemical structures of 1–4.
R1 R2
OH
H
2
1
26
OH
H
OCH3
3'
O
22 24
18
HO
HO
1
'''
OH
19
2'
H
O
27
4'
OH
H
8
H
H
H CO
9
O
2
H
O
3
7
5'
O
1'
HO
HO
3
6'
1
'
10
3
O
4
30
6
OH
H
H
OH
5
4
R
HO
H
H
H
H O
H O
29
28
HO
HO
O
1''
H
1
''
OH
HO
HO
H
OH
H
H
H
H
1
2
R = OH R = OH
3 R = H
4 R = OCH
3
1
2
R = H R = OH
1
2
This is the first report of cycloartane-type triterpenoids from the Lemnaceae family. Here the
isolation and structural elucidation of these compounds as well as bioactivities of compound 3
were described.

Molecules 2014, 19
6625
2
. Results and Discussion
2
.1. Structure Elucidation of Compounds 1–4
Compound 1 was obtained as an amorphous powder. Its HR-ESI-MS quasi-molecular ion peak at
+
m/z 985.5371 ([M+Na] ) corresponded to a molecular formula of C H O , which was supported by
4
8
82 19
1
the NMR signals. H-NMR spectrum of 1 showed characteristic signals of a cyclopropane CH
2
at δ
1.11, 1.24,
.23, 1.13, 0.95, 1.01; respectively, H -18, H -26, H -27, H -28, H -29, H -30) and a secondary Me
H
0
.64 (d, J = 3.72 Hz, H-19a) and 0.44 (d, J = 3.72 Hz, H-19b), six tertiary Me groups (δ
H
1
3
3
3
3
3
3
H 3 H
group at δ 0.95 (H -21) [17,18]. Additionally, three anomeric proton signals were observed at δ 4.74
(
d, J = 7.68 Hz), 4.52 (d, J = 7.20 Hz) and 4.48 (d, J = 7.74 Hz), indicative of the presence of three
1
3
β-linked sugar units. The C-NMR and DEPT spectra permitted differentiation of the 48 resonances
into 6 C, 22 CH, 13 CH , and 7 CH groups, of which 30 were attributed to a triterpene skeleton and 18
to three hexose groups. Acid hydrolysis of 1 gave D-glucoses with optical rotation of
2
3
2
D
0
[α
]
+ 51.2,
which was determined by TLC analysis and optical rotation measurement [19,20]. Therefore, 1 was
considered to be a cycloartane-type triterpene glucoside.
In the HMBC spectrum (Figure 2), correlations of δ 1.13 (H-28) and δ 0.95 (H-29) with δ 90.9
H
H
C
(C-3) suggested that C-3 was substituted by the OH group; 26-CH
3
, 27-CH
3
with C-24 (δ
C
76.2) and
C 3 C
C-25 (δ 74.0), and 21-CH with C-22 (δ 71.3) indicated the presence of 24-OH, 25-OH and 22-OH
respectively. The glycosidic linkage was determined on the basis of following key spectral signals:
1
1
HMBC correlations of H-1' with C-3, H-1'' with C-2', and H-1''' with C-6', H, H-COSY cross peaks of
H-1'/H-2', H-6'/H-5', and the NOESY cross peaks of H-1'/H-3' and H-5'. Furthermore, the
stereochemistry of C-24 was assigned to be S by comparing its spectral data with those reported for
analogs [21,22]. Additionally, NOESY correlations between δ
CH , 18-CH and δ 1.79 (H-20), δ 0.44 (H-19a) and 29-CH
2)/30-CH , 21-CH and δ 1.80 (H-17), H-17 and 30-CH were observed, revealing their relative
configuration (Figure 3).
H
1.62 (H-8) and δ
H
0.44 (H-19a)/18-
3
3
H
H
3
, δ 2.04 (H-16b) and δ
H
H
4.01 (H-
2
3
3
H
3
Figure 2. Key HMBC correlations of 1 and 2.
OH OH
OH
OH
OH
H
H
H
H
O
O
OH
OH
HO
HO
HO
HO
O
H
O
H
H
H
OH
OH
H
H
H
H
H
H
H O
O
H O
O
HO
HO
HO
HO
O
O
OH
H
OH
H
H
H
H
H
H
H O
O
HO
HO
HO
HO
H
OH
H
OH
H
H
H
H
Consequently, the structure of 1 was established as (3β,24S)-9,19-cycloartane-3,22,24,25-tetraol 3-
O-[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→6)]-β-D-glucopyranoside.
+
The HR-ESI-MS spectrum of 2 (m/z 969.5406 [M + Na] , calc. for C48
H
82
O18Na) supported a
molecular formula of C H O . The NMR spectra of 2 were very similar to those of 1, except for one
4
8
82 18
C
less oxygenated methine at δ 71.3. Detailed inspection of the HMBC spectrum led to a conclusion

Molecules 2014, 19
6626
that 2 had one less OH on C-22 than 1, on the basis of the key HMBC correlation of 21-CH (δ 1.01)
3
H
C
with C-22 (δ 23.2). The linkage of three glucoses on the triterpenoid aglycon was determined to be
1
1
the same way as 1 on the basis of HMBC and H, H-COSY signals. The stereochemistry of 2 was
determined on the basis of NOESY cross peaks of H-8 and H-19a/18-CH , 18-CH and H-12a, H-19a
3
3
3 3 3 3
and 29-CH , H-16b and 30-CH , 21-CH and H-17, H-17 and 30-CH . Acid hydrolysis of 2 gave
D-glucoses, which was identified by comparing the optical rotation value with an authentic sample.
Finally the structure of 2 was determined to be (3β,24S)-9,19-cycloartane-3,24,25-triol 3-O-[β-D-
glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→6)]-β-D-glucopyranoside.
Figure 3. Key NOE correlations of 1 and 2.
Compound 3 was obtained as colorless needles. Its molecular formula was deduced to be C H O
28 11
2
3
−
from the quasi-molecular ion peak at m/z 479.1549 ([M–H] ) in the HR-ESI-MS spectrum, indicating
−
1
1
0 degrees of unsaturation. The IR spectrum revealed the existence of OH (3434 cm ) and aromatic
−1
1
ring (1623 cm ). The H-NMR spectrum displayed two meta-coupled protons at δ
and 6.29 (d, J = 2.16 Hz), and three ABX system aromatic protons at δ 7.22 (d, J = 1.06 Hz), 6.99
dd, J = 8.16, 1.06 Hz), and 6.87 (d, J = 8.16 Hz). In addition, two methoxyl H-atoms at δ 3.82 (s,
3.09 (d, J = 17.60 Hz,
H) and 3.02 (dd, J = 17.60, 4.38 Hz, 1H) were observed. Meanwhile an anomeric H-atom at δ 4.95
3.42-3.55 indicated the presence of a β-linked glycosyl
H
6.49 (d, J = 2.16 Hz)
H
(
H
H 2 H
3H) and 3.95 (s, 3H), two CH at δ 4.29 (br. s) and 4.98 (br. s), and one CH at δ
1
H
(
d, J = 7.20 Hz) together with the signals at δ
H
group. Enzymatic hydrolysis of 3 with β-D-glucosidase afforded D-glucose, which was identified by
direct comparison with the authentic sample by TLC [19]. In view of above evidences, it was
concluded that 3 was a flavanol glucoside.
1
3
In the combination of the C-NMR and HSQC spectra of 3, the 23 carbon resonances could be
easily attributed to a flavanol moiety, a glucopyranose unit (δ 62.7, 71.6, 75.1, 78.2, 78.4, 102.8), and
C
two methoxyls (δ
moiety, HMBC and NOESY experiments were performed. As a result, the NOESY correlations of the
methoxyl H-atoms at δ 3.82 with H-6 and H-8, H-6 with H-1', and the other methoxyl H-atoms at δ
.95 with H-2', together with the HMBC correlations of 7-OCH to C-7, 3'-OCH to C-3', indicated the
glucose and two methoxyls were situated at C-5, C-7, and C-3' respectively (Figures 4 and 5).
The NOESY correlation of δ 4.98 (H-2)/δ 4.29 (H-3) and resonance of H-2 as a broad singlet
C
56.0, 56.6). In order to determine the location of substituent groups on the flavanol
H
H
3
3
3
H
H
indicated that the relative configuration of 2,3 was cis [23–28]. Therefore, the structure of 3 was
elucidated to be cis-3,4'-dihydroxy-7,3'-dimethoxyflavan-5-O-β-D-glucopyranoside.
Compound 4 was isolated as amorphous powder. The molecular formula was C24
30 12
H O ,
determined by negative-ion at m/z 509.1662 in the HR-ESI-MS. Enzymatic hydrolysis of compound 4

Molecules 2014, 19
6627
with β-D-glucosidase gave D-glucose. Comparing its NMR spectra with those of 3, it was evident that
contained one more methoxyl at δ 3.65/δ 56.6 and one oxygenated CH (δ 73.5) than 3.
The location of one more methoxyl at C-4 was determined by the HMBC correlation of 4-OCH
δ 3.65 with C-4 (δ 73.5). The other substitutions of 4 were confirmed by the same way as 3.
4
H
C
C
3
at
H
C
Figure 4. Key HMBC correlations of 3 and 4.
OCH3
OH
OCH3
OH
H CO
3
O
H CO
3
O
OH
OH
H
H
OH
OH
OCH3
H
H
O
O
HO
HO
HO
HO
O
O
H
H
OH
OH
H
H
H
H
The configuration of 4 was elucidated on the basis of NOESY correlations and coupling constants.
Resonances of H-2 as a broad singlet and H-4 as a small doublet (J = 2.28 Hz) were observed, which
suggested that the relative 2,3/3,4-stereochemistry were both cis [26]. Additionally the NOESY
correlation between δ
H
5.05 (H-2)/δ
H
4.62 (H-4) also supported the above assignment. The structure of
4
was identified to be cis-3,4'-dihydroxy-4,7,3'-trimethoxyflavan-5-O-β-D-glucopyranoside.
Figure 5. Key NOE correlations of 3 and 4.
OCH3
OH
OCH3
OH
H CO
O
3
H CO
O
3
H
H
OH
H
H
OH
H
OH
H
OH
H
O
H
H
H
H
O
HO
HO
OCH3
O
HO
HO
O
H
OH
H
H
H
OH
H
H
2
.2. Biological Activity Assay
According to previous bioactivity reports on flavonoids [29–31], antimicrobial and antioxidant
activities of compound 3 were evaluated. Two pathogenic bacteria (Bacillus subtilis, Escherichia coli)
and two fungi (Aspergillus niger, Saccharomyces cerevisiae) were selected for antifungal and
antibacterial assays, respectively. The compound 3 showed no antimicrobial activity (C > 50 μg/mL).
In the ABTS cation radical scavenging assay, the inhibition rate of 3 was 82.0% at 1 mg/mL (Positive
control vitamin C, 75.2%, C = 0.1 mg/mL). The superoxide anion radical scavenging assay suggested
that 3 showed 53.5% superoxide anion radical scavenging capacity at 2 mg/mL. While the positive

Molecules 2014, 19
6628
control luteolin was 53.8% at 0.625 mg/mL. Unfortunately, the low available amount of compounds 1,
2
3
3
, and 4 precluded the antimicrobial and antioxidant assays.
. Experimental
.1. General Information
Column chromatography (CC): silica gel (SiO , 200–300 mesh, Qingdao Marine Chemical Plant,
2
Qingdao, P. R. China), MCI gel (Mitsubishi Chemical Corporation, Tokyo, Japan), and Sephadex LH-20
GE Healthcare Bio-Sciences AB, USA). TLC: silica gel GF254 (Qingdao Marine Chemical Plant,
(
Qingdao, P. R. China). UV absorbance of antioxide mixtures: Varioskan Flash Reader (ThermoFisher
Scientific Inc., Waltham, MA). UV spectra: Perkin-Elmer S2 Lambda 35 UV/VIS spectrometer, λ in
−1
nm. IR spectra: Perkin-Elmer Spectrum One FT-IR spectrometer, as KBr tablets, ν in cm . Optical
rotations: Perkin-Elmer 341 polarimeter. NMR spectra: Bruker Avance 600 MHz instrument, δ in ppm,
J in Hz, residual solvent peak as reference. HR-ESI-MS: BioTOF-Q mass spectrometer, in m/z.
3
.2. Material
The duckweed Landoltia punctata (G. Meyer) Les & Crawford was grown under natural conditions
and collected in Kunming, Yunnan province, P. R. China. The duckweed was collected in May,
washed with water, and then dried at 60 °C. The test strains Aspergillus niger, Saccharomyces
cerevisiae, Escherichia coli, and Bacillus subtilis were obtained from Chengdu Institute of Biology,
Chinese Academy of Sciences (CAS), P. R. China.
3.3. Extraction and Isolation
The powder (7 kg) of L. punctata was macerated in 95% ethanol (70 L) at room temperature for
4
–5 days, twice. The extract was combined and concentrated under reduced pressure. Then the
concentrate was suspended in hot water, and extracted with petroleum ether (P.E.), EtOAc, and
n-BuOH successively. The P.E. extract was separated by silica gel column chromatography (CC) using
P.E./acetone (10:1–1:1, v/v) as eluent, affording 4 fractions (Fr. 1-4), Fr. 2 and 3 were purified by
Sephadex LH-20 CC to yield compounds 15–17 respectively.
The EtOAc fraction (25 g) was subjected to a MCI column, and eluted with a stepwise gradient of
EtOH/H
purified by HPLC (ODS-C18 column, 10 × 250 mm, flow rate 2.5 mL/min). Fr. I was separated using
MeOH/H O (40:60, containing 0.5% acetic acid, v/v) as the eluent to yield compounds 11 and 12. Fr.
II was purified using MeOH-H O (45:55, containing 0.5% acetic acid, v/v) as the eluent to yield
2
O (0:100, 20:80, 40:60, 60:40, 100:0, v/v) to give three fractions (I-III), which were further
2
2
2
compounds 7–10. Fr. III was separated using MeOH-H O (60:40, containing 0.5% acetic acid, v/v) as
the eluent to obtain compounds 5 and 6.
The same CC of the n-BuOH extract (60 g) on MCI gel as that of EtOAc extract gave four fractions
I–IV). Fraction II was subjected to preparative thin layer chromatography (TLC) and Sephadex LH-20
(
CC to afford compounds 1 (13 mg) and 2 (10 mg). Faction III was separated over silica gel with
CHCl /MeOH (7:1–0:1, v/v) as eluent to generate 4 fractions (IIIa-IIId). Fr. IIIb was separated by
HPLC using MeOH/H O (45:55, v/v) as the eluent to yield compounds 13 and 14. Fr. IIIc and IIId
3
2

Molecules 2014, 19
6629
were further purified by TLC and then Sephadex LH-20 CC to yield compounds 3 (18 mg) and
4
(8 mg) respectively.
3
.4. Hydrolysis
Acid hydrolysis. Compounds 1 and 2 (10 mg) were independently dissolved in aqueous solution of
2
N HCl (6 mL) and stirred at 85 °C overnight. After cooled down, the reaction mixture was
neutralized and then extracted with CHCl . The water layer were concentrated to dryness, dissolved in
3
water to constant volume, and analyzed by comparing their TLC profile and optical rotation values
with the standard sample of D-glucose [21,22], respectively.
Enzymatic hydrolysis. Compounds 3 and 4 (5.0 mg) were independently suspended in water (5 mL),
and excessive β-D-glucosidase (Shanghai Zurui Biological Technology Co., Ltd., Shanghai, China)
was added, respectively. The mixture was placed in water bath at 37 °C for 48 h. The products were
analyzed by TLC [21].
3.5. Spectral Data
(
3β,24S)-9,19-Cycloartane-3,22,24,25-tetraol 3-O-[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranos-
20
yl(1→6)]-β-D-glucopyranoside (1): Amorphous powder.
2
[α
]
−3.0 (c 0.1, MeOH). IR (KBr) νmax 3411,
D
−1
1
13
+
932, 1075 cm . H and C-NMR: See Table 1. HR-ESI-MS (positive mode) m/z 985.5371 [M+Na]
(
82
calc. for C48H O19Na, 985.5343).
(
3β,24S)-9,19-Cycloartane-3,24,25-triol
3-O-[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-
20
(1→6)]-β-D-glucopyranoside (2): Amorphous powder.
[α
]
−4.0 (c 0.1, MeOH). IR (KBr) νmax 3418,
D
−1
1
13
+
2
891, 1043 cm . H and C-NMR: See Table 1. HR-ESI-MS (positive mode) m/z 969.5406 [M + Na]
(
calc. for C48
H
82
O18Na, 969.5393).
2
D
0
3
0
1
,4'-Dihydroxy-7,3'-dimethoxyflavan-5-O-β-D-glucopyranoside (3): Colorless needles.
[α
]
−42.0 (c
.1, MeOH). UV (MeOH) λmax (log ε) 218 (4.60), 227 (3.96), 282 (3.52) nm. IR (KBr) νmax 3434,
623, 1080 cm . H and C-NMR: See Table 2. HR-ESI-MS (negative mode) m/z 479.1549 (calc. for
−1
1
13
C H O , 479.1559).
2
3
27 11
2
D
0
3
,4'-Dihydroxy-4,7,3'-trimethoxyflavan-5-O-β-D-glucopyranoside (4): Amorphous powder.
[α
]
−20.0
(
c 0.1, MeOH). UV (MeOH) λmax (log ε) 226 (4.02), 280 (3.60) nm. IR (KBr) νmax 3420, 2926, 1597,
424, 1079 cm . H and C-NMR: See Table 2. HR-ESI-MS (negative mode) m/z 509.1662 (calc. for
−1
1
13
1
24 29
C H O12, 509.1664).
3
.6. Biological Activity Assay
According to the reported [32], antifungal and antibacterial activities were determined using the
Oxford cup method with Methicillin sodium as the positive control. The antioxidant activity assay was
+
•
performed on the DPPH, ABTS , and superoxide anion radical scavenging models complying with the
previously published literature [33].

Molecules 2014, 19
Table 1. H (600 MHz) and C (150 MHz) NMR data of compounds 1 (in CD OD, J in Hz) and 2 (in C D N, J in Hz).
6630
1
13
3
5
5
1
2
1
2
No.
No.
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
1
2
3
4
5
6
7
8
9
33.3
30.7
90.9
42.4
50.4
22.3
27.4
49.7
21.4
27.5
27.7
34.2
46.9
49.8
37.0
28.4
50.0
18.7
30.9
1.65–1.67 (m), 1.32–1.34 (m) (m)
2.08–2.11 (m), 1.35–1.36 (m)
3.34–3.36 (m)
30.3
29.3
89.1
41.7
47.8
27.2
33.5
53.3
20.2
26.9
21.5
32.6
45.9
49.3
36.7
28.8
48.4
19.9
30.0
2.50–2.53 (m), 1.25–1.26 (m)
1.81–1.82 (m), 1.95–1.97 (m)
3.45–3.48 (m)
25
26
27
28
29
30
1'
74.0
26.1
25.6
26.0
15.6
20.1
105.3
81.4
78.2
71.7
77.1
70.2
104.8
76.5
78.4
72.0
78.1
63.0
105.0
75.4
78.0
71.9
78.1
63.3
73.1
26.5
26.2
26.0
15.7
18.6
1.24 (s, 3H)
1.23 (s, 3H)
1.56 (s, 3H)
1.54 (s, 3H)
1.13 (s, 3H)
1.29 (s, 3H)
1.68–1.70 (m)
1.67–1.68 (m), 1.68–1.70 (m)
1.39–1.40 (m), 1.36–1.37 (m)
1.60–1.63 (m)
1.20–1.22 (m)
0.95 (s, 3H)
1.19 (s, 3H)
1.87–1.88 (m), 1.37 (overlapped)
1.50–1.51 (m), 1.53 (overlapped)
1.64–1.65 (m)
1.01 (s, 3H)
0.99 (s, 3H)
4.52 (d, J = 7.20, 1H)
3.62–3.63 (m)
3.61–3.62 (m)
3.39–3.40 (m)
3.52–3.54 (m)
105.2
83.5
77.3
72.0
77.2
70.4
106.3
75.6
78.3
71.8
78.6
63.0
105.7
75.5
78.8
71.1
78.4
63.1
4.90 (d, J = 7.56, 1H)
4.15–4.17 (m)
2'
3'
4.10–4.11 (m)
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
4'
4.20–4.21 (m)
1.70–1.72 (m), 2.10–2.11 (m)
1.45–1.46 (m), 0.70–0.72 (m)
5'
4.03–4.04 (m)
1.72, 1.76, overlapped
1.64–1.65 (m), 1.24 (overlapped)
6'
3.85–3.87 (m), 4.16–4.18 (br. d)
4.74 (d, J = 7.68, 1H)
3.28–3.29 (m)
4.29–4.31 (m), 4.82 (br. d)
5.35 (d, J = 7.62, 1H)
4.04–4.05 (m)
1''
2''
3''
4''
5''
6''
1'''
2'''
3'''
4'''
5'''
6'''
1.40–1.41 (m), 1.42–1.43 (m)
1.42–1.43 (m), 2.03–2.05 (m)
1.79–1.81 (m)
1.65–1.66 (m), 1.55 (overlapped)
1.37–1.38 (m), 1.63–1.64 (m)
1.43–1.44 (m)
3.60–3.61 (m)
4.24–4.25 (m)
3.26–3,27 (m)
4.28–4.29 (m)
3.30–3.31 (m)
3.90–3.92 (m)
1.11 (s, 3H)
0.84 (s, 3H)
3.71–3.72 (m), 3.93–3.94 (m)
4.48 (d, J = 7.74, 1H)
3.24–3.26 (m)
4.45–4.46 (m), 4.52–4.53 (m)
5.13 (d, J = 7.80, 1H)
4.05–4.06 (m)
0.44 (d, J = 3.72)
0.24 (d, J = 4.08)
0
.64 (d, J = 3.72)
1.78–1.79 (m)
0.95 (s, 3H)
0.48 (d, J = 4.08)
2
2
2
2
2
0
1
2
3
4
44.0
12.7
71.3
32.9
76.2
36.1
18.9
23.2
34.5
79.4
1.23–1.24 (m)
3.40–3.42 (m)
3.94–3.96 (m)
1.01 (s, 3H)
3.31–3.32 (m)
4.03–4.04 (m)
4.01 (br. d)
0.86–0.87 (m), 1.25 (overlapped)
1.83–1.85 (m), 1.68 (overlapped)
3.78–3.81 (m)
3.29–3.30 (m)
4.21–4.22 (m)
1.40–1.41 (m), 1.53–1.57 (m)
3.58–3.60 (m)
3.70–3.71 (m), 3.90–3.92 (m)
4.33–4.36 (m), 4.48–4.50 (m)

Molecules 2014, 19
6631
1
13
Table 2. H (600 MHz) and C (150 MHz) NMR data of compounds 3 and 4 (in CD OD, J in Hz).
3
3
4
No.
δ
C
δ
H
δ
C
δ
H
2
80.3
4.98 (br. s, 1H)
78.6
5.05 (br. s, 1H)
3
4
67.4
29.7
4.29 (br. s, 1H)
3.09 (d, J = 17.60, 1H)
69.2
73.5
4.04 (br. d, 1H)
4.62 (d, J = 2.28, 1H)
3
.02 (dd, J = 17.60, 4.38, 1H)
5
6
158.6
97.1
157.9
96.6
6.49(d, J = 2.10)
6.29 (d, J = 2.10)
6.54 (d, J = 1.80)
6.30 (d, J = 1.80)
7
8
160.9
96.5
162.9
96.3
9
1
1
157.3
104.0
132.3
160.4
104.4
131.6
0
'
2
3
4
5
'
'
'
'
112.1
148.8
147.2
115.9
7.22 (d, J = 1.06)
112.3
149.0
147.4
116.0
7.22 (d, J = 1.10)
6.87 (d, J = 8.16)
6.90 (d, J = 8.04)
6
1
'
120.8
102.8
6.99 (dd, J = 1.06, 8.16)
121.0
102.6
7.00 (dd, J = 1.10, 8.04)
''
4.95 (d, J = 7.20)
4.99 (d, J = 7.50)
2
3
4
''
''
''
75.1
78.4
71.6
3.50–3.55 (overlapped)
3.50–3.55 (overlapped)
3.47–3.48 (m)
75.3
78.5
71.6
3.58–3.59 (overlapped)
3.53–3.56 (overlapped)
3.46–3.48 (overlapped)
5
6
''
''
78.2
62.7
3.50–3.55 (overlapped)
3.79 (dd, J = 5.58, 12.10)
76.9
62.8
3.53–3.56 (overlapped)
3.79 (dd, J = 5.00, 12.20)
4.00 (d, J = 12.20)
3.65 (s, 3H)
3
.98 (d, J = 12.10)
MeO-4
MeO-7
56.6
56.0
56.6
56.0
3.82 (s, 3H)
3.95 (s, 3H )
3.84 (s, 3H)
MeO-3'
57.8
3.96 (s, 3H)
4
. Conclusions
In this study, we identified 12 flavonoids including two new flavanol glucosides from L. punctata.
The result showed that apigenin or luteolin flavonoids are the main constituents of this species, which
is in good agreement with the previous reports and our previous study results that the transcripts for
key enzymes of flavonoid biosynthesis in L. punctata expressed in high abundance at the
transcriptional level [9,10,34]. Flavonoids are generally biosynthesized to cope with environmental
stressors such as ultraviolet radiation, ozone, heavy metals, nutrient limitation, herbivores, and so on.
The high content of flavonoids in L. punctata could be related to environmental stressors. Meanwhile
cycloartane triterpenoids were discovered in Lemnaceae family for the first time in this study. Many
cycloartane triterpenoids possessed diverse bioactivities such as anti-inflammatory, anti-tumor,
anti-viral, immuno-regulatory, hypoglycemic, cardiovascular system, nervous system and hepato-
protective effects [35,36], some of which offer good prospectives in medical applications. This study
demonstrates that L. punctata is a new source for cycloartane triterpenoids.

Molecules 2014, 19
6632
Acknowledgments
This research was financially supported by the Major Projects of Knowledge Innovation Program of
Chinese Academy of Sciences (KSCX2-EW-J-22).
Author Contributions
N.N.W performed all phytochemical parts of the work and prepared a draft manuscript. T.Y and
G.B.X performed the biological activity assays. Y.F offered help on the plant material. H.Z and G.Y.L
initiated the study, coordinated the project, supervised the phytochemical work and prepared the
final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
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(