Four new flavonol glycosides from the leaves of Brugmansia suaveolens

Article abstract of DOI:10.3390/molecules19056727

Four new flavonol glycosides were isolated from the leaves of Brugmansia suaveolens: kaempferol 3-O-β-D-glucopyranosyl-(1′→2′)-O- α-L-Arabinopyranoside (1), kaempferol 3-O-β-D-glucopyranosyl- (1′→2′)-O-α-L-Arabinopyranoside-7-O-i-D- glucopyranoside (2), k

Full text of DOI:10.3390/molecules19056727

Molecules 2014, 19, 6727-6736; doi:10.3390/molecules19056727
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Four New Flavonol Glycosides from the Leaves of Brugmansia suaveolens
Fabiana Geller 1,*, Renato Murillo 2, Lisa Steinhauser 3, Berta Heinzmann 4, Klaus Albert 3,
Irmgard Merfort 5 and Stefan Laufer 1
1
Department of Pharmaceutical/Medicinal Chemistry, University of Tübingen, Tübingen 72076,
Germany
2
Escuela de Quimica and CIPRONA, University of Costa Rica, San José 2060, Costa Rica
3
Department of Organic Chemistry, University of Tübingen, Tübingen 72076, Germany
4
Department of Industrial Pharmacy, Federal University of Santa Maria, Santa Maria 97105-900, Brazil
5
Department of Pharmaceutical Biology and Biotechnology, University of Freiburg,
Freiburg im Breisgau 79104, Germany
* Author to whom correspondence should be addressed; E-Mail: fabiana.geller@ufsc.br;
Tel.: +55-48-9663-8306.
Received: 31 March 2014; in revised form: 16 May 2014 / Accepted: 19 May 2014 /
Published: 22 May 2014
Abstract: Four new flavonol glycosides were isolated from the leaves of Brugmansia
suaveolens: kaempferol 3-O-β-D-glucopyranosyl-(1'''→2'')-O-α-L-arabinopyranoside (1),
kaempferol
3-O-β-D-glucopyranosyl-(1'''→2'')-O-α-L-arabinopyranoside-7-O-į-D-gluco-
pyranoside (2), kaempferol 3-O-β-D-[6'''-O-(E-caffeoyl)]-glucopyranosyl-(1'''→2'')-O-α-L-
arabinopyranoside-7-O-β-D-glucopyranoside (3), and kaempferol 3-O-β-D-[2'''-O-(E-
caffeoyl)]-glucopyranosyl-(1'''→2'')-O-α-L-arabinopyranoside-7-O-β-D-glucopyranoside (4).
The structure elucidation was performed by MS, 1D and 2D NMR analyses.
Keywords: Brugmansia suaveolens; Solanaceae; acylated kaempferol glycosides
1. Introduction
Brugmansia suaveolens (Humb. & Bonpl. ex. Willd.) Bercht. & C. Presl (Syn. Datura suaveolens),
known also as angel’s trumpet, is a flowering shrub of the Solanaceae family, native to the coastal
rainforest regions of Southeast Brazil. Extracts from its leaves have been studied for their anti-
inflammatory and wound healing activities [1,2]. Up until now, alkaloids [3–5] and essential oils [6]

Molecules 2014, 19
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have been reported as the main effective compounds. Moreover, in [7] the isolation of the flavonol
glycosides kaempferol 3-O-α-L-arabinopyranoside and kaempferol 3-O-α-L-arabinopyranoside-7-O-β-
D-glucopyranoside from the leaves of this species has been described. In this paper, the isolation and
structure elucidation of four new kaempferol glycosides are reported for the first time.
2. Results and Discussion
Phytochemical investigation of the ethanolic extract of B. suaveolens, prepared from the leaves,
yielded four new kaempferol glycosides, while neither of the compounds described by [7] were
isolated in this work. Analysis of the spectroscopic data led to the identification of compounds 1–4
(Figure 1).
Figure 1. Chemical structures of compounds 1–4 isolated from the leaves of Brugmansia suaveolens.
5'
OH
6'
1'
4'
3'
8
O
R
1
7
6
9
2
3
2'
R1
R2
R3
OH
10
1''
5''
(1)
OH
OH
OH
5
4
O
3''
O
2''
4''
(2) O-Glc
OH
O
OH
O
OH
R
2
6'''
3'''
OH
(3) O-Glc O-Caffeoyl
(4) O-Glc OH
OH
4'''
O
5'''
HO
HO
O-Caffeoyl
2'''
1'''
R
3
The molecular formula of compound 1 was determined as C26H28O15 on the basis of FT-ICR-MS
m/z 603.132636 [M+Na]+ (calcd. m/z 603.13204 [M+Na]+) and ESI-MS m/z 603.1 [M+Na]+. The IR
spectrum showed the characteristic absorption bands of hydroxyl (3248.2 cm−1), carbonyl (1652.8 cm−1),
1
and phenyl groups (1574.4 cm−1). The H-NMR and 13C-NMR spectra (see Table 1) are similar to
those reported by [7]. The aromatic region of the 1H-NMR spectrum of 1 showed four signals, namely,
two broad singlets (δ 6.21 and δ 6.40) for the protons in ring A and two doublets (AA'BB' system) for
ring B (δ 6.92 and δ 8.02, both with J = 8.6 Hz), which are typical for a kaempferol aglycone [7]. The
middle region of the spectrum exhibited two anomeric signals due to the sugar units at δ 5.48 and δ
4.55. The coupling constant of the anomeric proton of the glucose (J = 7.8 Hz) was in accordance with
a β-glycosidic linkage, while the coupling constant of the anomeric proton of the pentose (J = 3.3 Hz)
indicated a α-glycosidic linkage [8]. Acid hydrolysis of 1 indicated D-glucose and L-arabinose, which
were compared with an authentic sample by TLC analysis [9,10]. A further eleven protons were
identified between δ 3.34 and δ 4.21 due to the sugar units [8]. The structural information concerning
the attachment position of the aforementioned moieties was determined based on MS fragmentation
and 2D-NMR analysis. The MS fragmentation of 1 showed no [M+H−pentose]+ fragment, indicating
that the pentose is bonded directly to the aglycone, and that the glucose unit is linked as the second sugar
moiety [11,12]. The HMBC experiment of 1 exhibited a correlation of the anomeric proton H-1'' of
α-L-arabinopyranose (δ 5.48) with C-3 (δC3 135.7), indicating the linkage of this sugar with the
aglycone moiety. The interglycosidic linkage is shown by the correlation of the H-1''' (δ 4.55) of

Molecules 2014, 19
6729
β-D-glucopyranose with the C-2'' (δC2'' 80.1) of α-L-arabinopyranose [13]. Based on the aforementioned
spectroscopic data, the structure of 1 was assigned as a kaempferol 3-O-β-D-glucopyranosyl-(1'''→2'')-
O-α-L-arabinopyranoside.
Table 1. 1H-NMR, 13C-NMR, and 2D-NMR spectral data for compounds 1 and 2.
1 a
2 b
Position
2
δH (J in Hz)
δC
COSY
HMBC
δH (J in Hz)
δC
COSY
HMBC
158.5
135.7
179.7
161.6
99.9
155.9
134.3
177.7
160.2
99.3
3
4
5
6
6.21
Brs
Brs
H-8
H-6
C-7,8,10 6.44
C-6,7,9,10 6.79
Brs
Brs
H-8
H-6
C-5,7,8,10
C-6,7,9,10
7
166.0
94.7
162.9
94.6
8
6.40
9
159.0
105.8
122.6
132.4
116.5
163.1
116.5
132.4
156.6
105.6
120.3
131.1
115.4
160.2
115.4
131.1
10
1'
2'
8.02
6.92
d (8.6)
d (8.6)
H-3'
H-2'
C-2,4'
8.10
6.91
d (8.4)
d (8.4)
H-3'
H-2'
C-2,4'
C-1',4'
3'
C-1′,4'
4'
5'
6.92
8.02
d (8.6)
d (8.6)
H-6′
H-5′
C-1',4'
C-2, 4'
6.91
8.10
d (8.4)
d (8.4)
H-6'
C-1',4'
C-2, 4'
6'
H-5′
Ara-O-3
1''
5.48
4.21
3.96
3.86
3.23
3.73
d (3.3)
101.3
H-2''
H-1'',3''
H-2'',4''
H-3'',5''
H-4′′
C-3
5.61
4.07
3.86
3.70
3.07
d (3.1)
M
98.9
78.7
68.7
64.1
61.2
H-2''
H-1'',3''
H-2'',4''
H-3'',5''
H-4''
C-3
2''
dd (5.4,3.4) 80.1
dd (5.4,6.4) 71.3
3′′
M
4''
M
M
M
66.6
63.4
M
5a''
5b′′
Glc-Ara
1'''
2'''
3'''
4'''
5'''
6a'''
6b'''
Glc-O-7
1''''
2''''
3''''
4''''
5''''
6a''''
6b''''
M
3.51 brd (11.1)
4.55
3.25
d (7.8)
105.4
75.2
78.1
71.4
78.0
62.7
H-2'''
H-1''',3'''
H-2''',4'''
H-3''',5'''
H-4''', 6'''
H-5'''
C-2''
4.37
2.97
3.17
3.12
3.43
d (7.5)
M
103.8
73.6
76.7
69.7
H-2'''
C-2''
brd (7.8)
H-1''',3'''
H-2''',4'''
H-3''',5'''
3.38
M
M
M
M
M
3.34
M
3.36
M
77.1 H-4''', 6'''
3.78–3.82
3.43 brd (11.5) 60.9
H-5′′′
3.59
M
5.07
3.25
3.30
3.17
3.12
3.70
3.43
d (7.1)
M
99.8
H-2''''
C-7
73.1 H-1'''',3''''
76.4 H-2'''',4''''
69.6 H-3'''',5'''',
76.8 H-4'''',6''''
M
M
M
M
60.6
H-5''''
M
a MeOH-d4; b DMSO-d6; 13C-NMR measured in 100 MHz; 1H-NMR measured in 600 MHz.

Molecules 2014, 19
6730
Compound 2 was established as C32H38O20 on the basis of FT-ICR-MS m/z 765.184176 [M+Na]+
(calcd. m/z 765.18486 [M+Na]+) and ESI-MS m/z 765.0 [M+Na]+. The IR spectrum revealed signals of
hydroxyl (3365.8 cm−1), carbonyl (1653.8 cm−1), and phenyl groups (1605.3 cm−1). The 1H-NMR and
13C-NMR (see Table 1) spectra are similar to those of 1. In the middle region of the spectrum, one
additional anomeric signal was identified, due to a new sugar unit at δ 5.07. The coupling constant of
this anomeric proton (J = 7.1 Hz) was in accordance with a β-glycosidic linkage. The sugar units of
compound 2 were determined as D-glucose and L-arabinose by TLC comparison with authentic
samples after acid hydrolysis [9,10]. As in 1, the HMBC experiment of 2 exhibited correlation between
the anomeric proton H-1'' (δ 5.61) and C-3 (δC3 134.3), and between H-1'''' (δ 5.07) and C-7 (δC7
162.9), indicating, respectively, the linkage of α-L-arabinopyranose and of β-D-glucopyranose with the
aglycone. Additionally, the interglycosidic linkage is shown by the correlation of the H-1''' (δ 4.37) of
another β-D-glucopyranose with the C-2'' (δC2'' 78.7) of α-L-arabinopyranose [13]. Therefore,
compound 2 was identified as a kaempferol 3-O-β-D-glucopyranosyl-(1'''→2'')-O-α-L-arabino-
pyranoside-7-O-β-D-glucopyranoside.
The molecular formula C41H44O23 of compound 3 was deduced based on FT-ICR-MS m/z
927.215977 [M+Na]+ (calcd. m/z 927.21656 [M+Na]+) and ESI-MS m/z 927.7 [M+Na]+. The
absorption bands of the hydroxyl (3309.5 cm−1), carbonyl (1651.9 cm−1), and phenyl groups (1598.2 cm−1)
13
were observed in IR analysis. The 1H-NMR and C-NMR spectra (see Table 2) were similar to those
of 2, including again three anomeric signals. Acid hydrolysis of 3 suggests D-glucose and
L-arabinose as sugar moieties, which was confirmed by comparison with authentic samples by TLC
analysis [9,10]. Additionally, 3 showed two further signals at δ 6.41 and δ 7.81 (both d, J = 15.8 Hz),
which are indicative of olefinic protons of a trans-caffeoyl group [14,15]. The HMBC experiment
exhibited correlation between the anomeric proton H-1'' of α-L-arabinopyranose (δ 6.41) and C-3 (δC3
137.0), and between the anomeric proton H-1'''''' (δ 5.78) and C-7 (δC7 163.6), showing direct linkages
of these sugars moieties with aglycone. The correlation of the H-2'' (δ 5.07) of α-L-arabinopyranose
with the C-1''' (δC1''' 106.7) of another β-D-glucopyranose indicates a linkage between these sugar
1
moieties. The H-NMR spectrum of 3 exhibited the downfield shift of the signals corresponding to
methylene protons H2-6''' to δ 4.90–5.03, indicating an acylation at this position [14]. The HMBC
experiment confirmed the linkage of the caffeic acid to C-6''' of β-D-glucopyranose by a correlation
between the H-6a''' and H-6b''' (δ 4.90–5.03) of this sugar unit and C=O (δC-1'''' 167.4) of the caffeic acid
moiety. Based on detailed analysis of the NMR spectra, the structure of compound 3 was determined
as a kaempferol 3-O-β-D-[6'''-O-(E-caffeoyl)]-glucopyranosyl-(1'''→2'')-O-α-L-arabinopyranoside-7-O-β-D-
glucopyranoside.
Compound 4 was determined as C41H44O23 based on FT-ICR-MS m/z 927.216170 [M+Na]+ (calcd.
m/z 927.21656 [M+Na]+) and ESI-MS m/z 927.1 [M+Na]+. The IR spectrum showed bands of
1
13
hydroxyl (3263.2 cm−1) and phenyl groups (1586.6 cm−1). The H-NMR and C-NMR spectra (see
Table 2) are similar to those of 3. Compound 4 showed again three anomeric signals and also two
further signals at δ 6.25 and δ 7.47 (both d, J = 15.7 Hz), which were assigned to the olefinic protons
of a trans-caffeoyl group [14,15]. Acid hydrolysis of 4, followed by TLC comparison with authentic
samples, indicated the presence of D-glucose and L-arabinose [9,10]. The HMBC experiment of 4
exhibited correlation between the anomeric proton H-1'' of α-L-arabinopyranose (δ 5.58) and C-3 (δC3
134.5), and between the anomeric proton H-1'''''' of a β-D-glucopyranose unit (δ 5.06) and C-7 (δC7 162.9),

Molecules 2014, 19
6731
showing the linkages of these sugar units to the kaempferol moiety. The interglycosidic linkage is
shown by the correlation of H-1''' (δ 4.68) of another β-D-glucopyranose with C-2'' (δC2'' 78.7) of
1
α-L-arabinopyranose [13]. The H-NMR spectrum of 4 exhibited also a downfield shift of the signal
corresponding to H-2''' at δ 4.60, indicating an acylation at the C-2''' position. The HMBC experiment
confirmed the assignment of the caffeic acid moiety to C-2''' of β-D-glucopyranose by a correlation
between the H-2''' (δ 4.60, t, J = 8.7 Hz) of this sugar unit and the C=O (δC-1'''' 165.6) of the caffeic
acid moiety.
Table 2. 1H-NMR, 13C-NMR, and 2D-NMR spectral data for compounds 3 and 4.
3 c
4 b
δH
(J in Hz)
δH
(J in Hz)
Position
δC
COSY
HMBC
δC
COSY
HMBC
2
157.2
137.0
178.8
161.7
100.0
163.6
94.6
154.4
134.5
177.6
160.5
98.7
3
4
5
6
6.72
brs
brs
H-8
H-6
C-5,7,8,10 6.43
C-6,7,9,10 6.76
Brs
Brs
H-8
H-6
C-7
7
162.9
94.0
8
6.92
C-7,10
9
156.6
106.7
121.8
131.8
116.2
162.0
116.2
131.8
155.9
106.0
121.2
131.0
115.4
160.5
115.4
131.0
10
1'
2'
8.44
7.26
d (8.6)
d (8.6)
H-3'
H-2'
C-2,4'
C-1',4'
8.06
6.81
d (8.6)
d (8.6)
H-3'
H-2'
C-2
3'
C-1',4'
4'
5'
7.26
8.44
d (8.6)
d (8.6)
H-6'
H-5'
C-1',4'
C-2,4'
6.81
8.06
d (8.6)
d (8.6)
H-6'
H-5'
C-1',4'
C-2
6'
Ara-O-3
1''
6.41
5.07
4.67
4.47
4.38
d (5.0)
100.4
80.7
70.9
66.0
62.1
H-2''
H-1'',3''
H-2'',4''
H-3'',5''
H-4''
C-3
5.58
4.09
3.85
3.50
d (3.4)
M
98.7
78.7
68.6
63.7
60.6
H-2''
H-1'',3''
H-2'',4''
H-3'',5''
H-4''
C-3
2''
m
m
m
m
3''
M
4''
M
5a''
5b''
Glc-Ara
1'''
2'''
3'''
4'''
5'''
3.00 brd (11.6)
4.56 brd (11.9)
3.50
M
5.28
4.12
4.05
4.15
4.05
4.90–
5.03
d (7.5)
106.7
75.1
78.1
70.9
75.4
H-2'''
C-2''
4.68
4.60
3.48
3.25
3.30
3.50–
3.70
d (7.8)
M
101.5
73.1
73.6
69.5
76.4
H-2'''
C-2''
m
m
m
m
H-1''',3'''
H-2''',4'''
H-3''',5'''
H-4''',6'''
H-1''',3'''
H-2''',4'''
H-3''',5'''
H-4''',6'''
C-1''''
M
M
M
6a'''
m
63.9
H-5'''
C-1''''
C1'''''
M
60.3
H-5'''
6b'''
Caffeoyl
1''''
167.4
165.6
113.9
2''''
6.41 d (15.8) 114.6
H-3''''
6.25
d (15.7)
H-3''''
C1'''''

Molecules 2014, 19
6732
Table 2. Cont.
3 c
4 b
δH
δH
Position
δC
COSY
HMBC
δC
COSY
HMBC
(J in Hz)
(J in Hz)
3''''
1'''''
7.81 d (15.8) 145.6
126.6
H-2''''
C-1′′′′, 2''''' 7.47 d (15.7)
145.0
125.4
114.9
145.7
148.7
115.8
H-2''''
C-1'''', 2'''''
C-3''''
2'''''
7.40
brs
115.6
145.6
147.2
C-3''''
7.07
6.90
Brs
3'''''
4'''''
5'''''
7.12 d (8.0) 116.3
6.95 d (8.0) 121.8
H-6'''''
H-5'''''
d (8.6)
H-6'''''
H-5'''''
6'''''
6.96 brd (8.0) 121.2
Glc-O-7
1''''''
2''''''
3''''''
4''''''
5''''''
5.78 d (7.2) 101.3
H-2''''''
C-7
5.06
3.20
3.28
3.25
3.40
3.50-
3.70
d (7.4)
M
99.8
72.5
76.8
69.8
77.1
H-2''''''
C-7
4.30
4.40
4.30
4.30
m
m
m
m
74.6
78.9
71.0
78.1
H-1'''''',3''''''
H-2'''''',4''''''
H-3'''''',5''''''
H-4'''''',6''''''
H-1'''''',3''''''
H-2'''''',4''''''
H-3'''''',5''''''
H-4'''''',6''''''
M
M
M
6a''''''
6b''''''
3.71
4.50
m
m
63.4
H-5''''''
M
60.3
H-5''''''
b DMSO-d6; c Pyridine-d5; 13C-NMR measured in 100 MHz; 1H-NMR measured in 600 MHz.
Therefore, the structure of 4 was assigned as a kaempferol 3-O-β-D-[2'''-O-(E-caffeoyl)]-
glucopyranosyl-(1'''→2'')-O-α-L-arabinopyranoside-7-O-β-D-glucopyranoside.
3. Experimental Section
3.1. General
13
NMR spectra 1D (1H, C, and DEPT-135) and 2D (1H-1H COSY, HSQC, HMBC) were recorded
with MeOH-d4, DMSO-d6 or pyridine-d5 on Bruker AMX-600 (600 MHz and 150 MHz) and Bruker
AMX-400 (400 MHz and 100 MHz) spectrometers (Bruker BioSpin GmbH, Rheinstetten, Germany).
LC-ESI-MS was carried out on an electrospray Finnigan MAT P4000 HPLC-DAD system connected
to a Finningan LCQTM Duo ion Trap mass spectrometer (Thermo Electron GmbH, Karlsruhe,
Germany). Analytical HPLC was performed with a L-7100 Pump (Merck-HITACHI-LaChrom); UV-Vis
detector L7420 (Merck-HITACHI-LaChrom); HPLC D-7000 HSM software with a D7000 data
interface. HR-MS (FT-ICR) was performed on a Bruker APEXII (electrospray ionization). The UV
spectra were measured in a Hewlett-Packard HP 1090 HPLC-DAD system in MeOH. The IR spectra
were obtained on a Perkin Elmer Spectrum One (ATR Technology, Shelton, CT, USA). Sephadex LH-20
(6 × 50 cm) (particle size 25–100 mm, Sigma Chemical Co., Munich, Germany); Thin layer
chromatography (TLC) (Silica Gel 60 F254 (0.25 mm) Merck (Darmstadt, Germany); RP-TLC
aluminum sheets 20 × 20 cm, RP-18 F254 Merck); Flash chromatography (LaFlash, VWR International,
Darmstadt, Germany), RP-18 (25–40 μm) column (5 × 20 cm), with gradient MeOH–H2O (10:90 
100:0, v/v); Analytical HPLC, LiChrospher RP-18 column (5 × 100 mm; 5 µm), mobile phase (A)
MeOH–ACN–FA (95:5:0.1, v/v/v) and (B) gradient of MeOH–ACN–FA (95:5:0.185:15:0.1, v/v/v).

Molecules 2014, 19
6733
3.2. Plant Material
Leaves of Brugmansia suaveolens were collected in Santa Maria, Rio Grande do Sul, Brazil in
January 2008. The plant was identified by botanist Gilberto Zanetti. A voucher specimen was
deposited in the herbarium of the Department of Biology at the Federal University of Santa Maria,
Brazil, under reference number SMDB12520.
3.3. Extraction and Isolation
The air-dried and powdered leaves (1.087 g) of B. suaveolens were exhaustively extracted with
EtOH (7 L, 30 h) in a Soxhlet apparatus. The EtOH extract was concentrated under vacuum at 40 °C
and lyophilized to yield 359.94 g, which was treated with MeOH at −20 °C giving a soluble fraction of
344.82 g after solvent removal. Of this extract, 6.18 g was subjected to column chromatography with
Sephadex LH-20 and MeOH as mobile phase at a flow rate of 1 mL/min. A total of 300 fractions of
10 mL each were collected and controlled by TLC using silica gel with toluene–MeOH–DEA (8:1:1,
v/v/v) and RP-18 with MeOH–H2O (1:1, v/v); Anisaldehyde-H2SO4 was used for detection. Those
fractions with a similar profile were combined, yielding 11 fractions (AK). Fraction G (80 mg) was
chromatographed by RP-18 CC using MeOH–H2O (1:1, v/v) to give five combined sub-fractions
(G1.1-G1.5). A sub-fraction G1.2 was fractioned again by RP-18 CC with MeOH–H2O (1:2, v/v) to
provide three sub-fractions (G1.2.1-G1.2.3). Sub-fraction G1.2.1 was further purified by HPLC using a
gradient of MeOH–ACN–FA (95:5:0.185:15:0.1, v/v/v), yielding compound 2 (10.3 mg). Fraction I
(41.1 mg) was chromatographed by RP-18 gel CC using MeOH–H2O (1:1, v/v) to give three combined
sub-fractions (I1.1-I1.3). Sub-fraction I1.2.2 was further purified by HPLC using a gradient of MeOH–
ACN–FA (95:5:0.185:15:0.1, v/v/v) to afford compound 3 (4.5 mg). Fraction H (100 mg) was
separated by flash chromatography RP-18 with gradient MeOH–H2O (10:90  100:0, v/v) to give four
combined sub-fractions (H1.1-H1.4). Sub-fraction H1.4 (47.8 mg) was further purified by RP-18 CC
using MeOH–H2O (1:1, v/v) yielding 3.2 mg of compound 1. Sub-fraction H1.2 (35.2 mg) was applied
to HPLC using a gradient of MeOH–ACN–FA (95:5:0.185:15:0.1, v/v/v), affording 11.1 mg of
compound 3 and 3.5 mg of compound 4.
3.4. Acid Hydrolysis
An amount of 2 mg per compound 1–4 was dissolved in EtOH–HCl 10% (10 mL) and refluxed at
80 °C for 2 h. The mixture was diluted in water (10 mL) and extracted with EtOAc (3 × 3 mL). The
aglycone and sugar moieties were identified by TLC analysis and compared with authentic samples,
D(+)-glucose, L(+)-arabinose, and kaempferol used as standards (Sigma-Aldrich, Munich, Germany).
Silica gel (Merck), mobile phase for aglycone CHCl3–EtOAc–MeOH (14:3:3, v/v/v) detected with
AlCl3, and mobile phase for sugars EtOH 96%–NH4OH 25%–H2O (20:1:4, v/v/v) detected with aniline
phthalate [9,10].
Kaempferol 3-O-β-D-glucopyranosyl-(1'''→2'')-O-α-L-arabinopyranoside (1). Yellow amorphous
powder. ESI-MS: positive ions m/z (rel. int.): 603.1 [M+Na]+ (54); 581.0 [M+H]+ (52); 419.0
[M+H−glucose]+ (43); 401.1 [M+H−glucose−H2O]+ (13); 287.2 [aglycone+H]+ (100). FT-ICR-MS

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(ESI): [Measured: 603.132636]+ (calculated mass for C26H28O15Na+: 603.13204). UV λ max (nm)
MeOH: 265, 346. 1H-NMR, 13C-NMR, COSY, and HMBC: Table 1.
Kaempferol
3-O-β-D-glucopyranosyl-(1′′′→2′′)-O-α-L-arabinopyranoside-7-O-β-D-glucopyranoside
(2). Yellow amorphous powder. ESI-MS: positive ions m/z (rel. int.): 765.0 [M+Na]+ (10); 742.8
[M+H]+ (22); 580.9 [M+H−glucose]+ (29); 448.8 [M+H−arabinose−glucose]+ (100); 418.9
[M+H−glucose−glucose]+ (100); 400.9 [M+H−glucose−glucose−H2O]+ (7); 287.1 [aglycone+H]+ (82).
FT-ICR-MS (ESI): [Measured: 765.184176]+ (calculated mass for C32H38O20Na+: 765.18486). UV λ
max (nm) MeOH: 265, 345. 1H-NMR, 13C-NMR, COSY, and HMBC: Table 1.
Kaempferol 3-O-β-D-[6′′′-O-(E-caffeoyl)]-glucopyranosyl-(1′′′→2′′)-O-α-L-arabinopyranoside-7-O-β-
D-glucopyranoside (3). Yellow amorphous powder. ESI-MS: positive ions m/z (rel. int.): 927.7
[M+Na]+ (13); 904.8 [M+H]+ (74); 742.9 [M+H−caffeic acid]+ (21); 580.8 [M+H−caffeic
acid−glucose]+ (24); 448.9 [M+H−caffeic acid−arabinose−glucose]+ (100); 418.8 [M+H−caffeic
acid−glucose−glucose]+ (7); 324.8 [caffeic acid+glucose−H]+ (8); 287.1 [aglycone+H]+ (37); 162.9
[caffeic acid−OH]+ (8). FT-ICR-MS (ESI): [Measured: 927.215977]+ (calculated mass for
1
C41H44O23Na+: 927.21656). UV λ max (nm) MeOH: 265, 328. H-NMR, 13C-NMR, COSY, and
HMBC: Table 2.
Kaempferol 3-O-β-D-[2′′′-O-(E-caffeoyl)]-glucopyranosyl-(1′′′→2′′)-O-α-L-arabinopyranoside-7-O-β-
D-glucopyranoside (4). Yellow amorphous powder. ESI-MS: positive ions m/z (rel. int.): 927.1
[M+Na]+ (42); 904.9 [M+H]+ (26); 742.9 [M+H−caffeic acid]+ (10); 581.0 [M+H−caffeic
acid−glucose]+ (14); 448.9 [M+H−caffeic acid−arabinose−glucose]+ (49); 419.1 [M+H−caffeic
acid−glucose−glucose]+ (6); 324.9 [caffeic acid+glucose−H]+ (25); 287.2 [aglycone+H]+ (100); 162.9
[caffeic acid−OH]+ (24). UV λ max (nm) MeOH: 265, 330. FT-ICR-MS (ESI): [Measured:
1
927.216170]+ (calculated mass for C41H44O23Na+: 927.21656). H-NMR, 13C-NMR, COSY, and
HMBC: Table 2.
4. Conclusions
The four kaempferol glycosides isolated from the leaves of B. suaveolens are described for the first
time in Nature. The similarities of the chemical structures suggest a common biosynthetic pathway
with the constituents reported by [7] for the same plant species. Additional studies considering their
biosynthesis should be performed to clarify this issue.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/5/6727/s1.
Acknowledgments
We thank Mrs. Ledi Marchi for helping to collect plant material, and botanist Gilberto Zanetti of the
Department of Industrial Pharmacy, Federal University of Santa Maria, Brazil, for the localization and
identification of the species. We acknowledge the Department of Organic Chemistry, University of

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Tübingen, Germany for running the NMR spectra. This work was supported by the government of
Baden-Württemberg (Zukunftsoffensive IV “Förderung von Innovation und Exzellenz”).
Author Contributions
Fabiana Geller was mainly responsible for the plant collection and extraction, isolation, structure
elucidation, and writing the article. Renato Murillo was in charge of interpreting the NMR spectra.
Lisa Steinhauser and Klaus Albert were responsible for providing the facilities for NMR analysis.
Berta Heinzmann was the main person responsible for plant collection and extraction, and also
performed a critical revision of the article. Irmgard Merfort was involved in the design of the project,
the structure elucidation and the critical revision of the article. Stefan Laufer was responsible for
obtaining funding, for the design of the project and for the final approval of the article.
Conflicts of Interest
The authors declare no conflict of interest.
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