Four new triterpenoids from callicarpa kwangtungensis

Article abstract of DOI:10.3390/molecules20059071

Four new triterpenoids which were identifed as 2α,3β,6β,19α-Tetrahydroxyoleanolic acid 28-O-β-D-glucopyranoside (1), 2-O-β-D-glucopyranosyloxy-3α,19α-dihydroxyoleanolic acid (2), 2-O-β-D-glucopyranosyloxy-3α,19α-dihydroxyursolic acid (3), 2α,3α,6β,19α-Tetrahydroxyursolic acid 28-O-β-D-glucopyranoside (4), were isolated from the aerial parts of Callicarpa kwangtungensis together with three known triterpenoids identified as 2α,3β,21β-Trihydroxyursolic acid 28-O-β-D-glucopyranoside (5), 2α,3α,19α,23-tetrahydroxyoleanolic acid 28-O-β-D-glucopyranoside (6), 2α,3α,19α,23-Tetrahydroxyursolic acid 28-O-β-D-glucopyranoside (7). Their structures were elucidated by the combination of mass spectrometry (MS), one and two-dimensional NMR experiments.

Full text of DOI:10.3390/molecules20059071

Molecules 2015, 20, 9071-9083; doi:10.3390/molecules20059071
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Four New Triterpenoids from Callicarpa kwangtungensis
1
1,3
1,3
2,
1
Guo-Ping Zhou , Yan Yu , Ming-Ming Yuan , Tengfei Ji *, Hui-Zheng Fu and
1
,
Rui-Jian Zhong *
1
Jiangxi Provincial Institute for Drug and Food Control, Nanchang 330029, China;
E-Mails: 13870868633@139.com (G.-P.Z.); 15070029890@163.com (Y.Y.);
1
3426688499@163.com (M.-M.Y.); fhzfhz620@sohu.com (H.-Z.F.)
2
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing 100050, China
3
Pharmaceutical Department of Nanchang University, Nanchang 330006, China
*
Authors to whom correspondence should be addressed; E-Mails: jitf@imm.ac.cn (T.J.);
jxfdc_zrj@126.com (R.-J.Z.); Tel.: +86-10-6012-2117 (T.J.); Fax: +86-10-6301-7757 (T.J.).
Academic Editor: Derek J. McPhee
Received: 1 March 2015 / Accepted: 11 May 2015 / Published: 19 May 2015
Abstract: Four new triterpenoids which were identifed as 2α,3β,6β,19α-tetrahydroxy-
oleanolic acid 28-O-β-D-glucopyranoside (1), 2-O-β-D-glucopyranosyloxy-3α,19α-di-
hydroxyoleanolic acid (2), 2-O-β-D-glucopyranosyloxy-3α,19α-dihydroxyursolic acid (3),
2α,3α,6β,19α-tetrahydroxyursolic acid 28-O-β-D-glucopyranoside (4), were isolated from the
aerial parts of Callicarpa kwangtungensis together with three known triterpenoids identified
as 2α,3β,21β-trihydroxyursolic acid 28-O-β-D-glucopyranoside (5), 2α,3α,19α,23-
tetrahydroxyoleanolic acid 28-O-β-D-glucopyranoside (6), 2α,3α,19α,23-tetrahydroxyursolic
acid 28-O-β-D-glucopyranoside (7). Their structures were elucidated by the combination of
mass spectrometry (MS), one and two-dimensional NMR experiments.
Keywords: Callicarpa kwangtungensis; Verbenaceae; triterpenoids; NMR; MS
1
. Introduction
Callicarpa kwangtungensis Chun, belonging to the family Verbenaceae, is distributed widely in the
Guangdong, Guangxi, and Jiangxi provinces of China [1]. The aerial parts of C. kwangtungensis are

Molecules 2015, 20
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used in Chinese herbal medicine for the treatment of bleeding wounds and hematemesis [1]. Previous
phytochemical studies of the genus Callicarpa led to the isolation of flavonoids, triterpenoids, and
phenylpropanoid glycosides. Some sesquiterpenoids such as callicarpenal, intermedeol, α-humulane
were isolated from Callicarpa americana, C. japonica and C. pedunculata; They also include a few
diterpenoids such as 16,17-dihydroxy-3-oxophyllocladane, 16-hydroxy-17-acetoxy-3-oxophyllocladane,
isopropylidenocalliterpenone, and calliphyllin from C. acuminata, C. formosana, C. macrophylla,
C. maingayi and C. pentandra; and many triterpenoids such as 2a,3a,24-trihydroxyoleanolic acid,
ursolic acid and β-amyrin from C. formosana; oleanolic acid, betulin and α-amyrin from
C. macrophylla; maslinic acid, bauerenol, 2α,3β-dihydroxyursolic acid from C. bodinieri; several
phenylpropanoid glycosides from C. pentandra, C. kwangtungensis and C. furfuraceae [2–4]. In our
previous investigations, a novel phenylpropanoid glycoside was isolated from the aerial parts of
C. kwangtungensis [5,6].
1
3
5
2
4
6
7
Figure 1. Structures of compounds 1–7.
As part of a study of the chemical constituents of C. kwangtungensis, a 95% EtOH extract of the
aerial parts of C. kwangtungensis has now been investigated. Four new triterpenoids have been isolated
and identified as 2α,3β,6β,19α-tetrahydroxyoleanolic acid 28-O-β-D-glucopyranoside (1), 2-O-β-D-

Molecules 2015, 20
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glucopyranosyloxy-3α,19α-dihydroxyoleanolic
acid
(2),
2-O-β-D-glucopyranosyloxy-3α,19α-
dihydroxyursolic acid (3), 2α,3α,6β,19α-tetrahydroxyursolic acid 28-O-β-D-glucopyranoside (4), In
addition three known triterpenoids identified as 2α,3β,21β-trihydroxyursolic acid 28-O-β-D-
glucopyranoside (5), 2α,3α,19α,23-tetrahydroxyoleanolic acid 28-O-β-D-glucopyranoside (6) and
2α,3α,19α,23-tetrahydroxyursolic acid 28-O-β-D-glucopyranoside (7) have also been isolated (Figure 1).
We report herein the isolation and structure elucidation of these compounds.
2
. Results and Discussion
The EtOH extract of the aerial parts of Callicarpa kwangtungensis was successively partitioned
with petroleum ether, EtOAc, and n-BuOH. The EtOAc-soluble portion was separated by a
combination of silica gel, ODS column chromatography, and preparative HPLC to afford four new
triterpenoids: 2α,3β,6β,19α-tetrahydroxyoleanolic acid 28-O-β-D-glucopyranoside (1), 2-O-β-D-
glucopyranosyloxy-3α,19α-dihydroxyoleanolic
acid
(2),
2-O-β-D-glucopyranosyloxy-3α,19α-
dihydroxyursolic acid (3), 2α,3α,6β,19α-tetrahydroxyursolic acid 28-O-β-D-glucopyranoside (4),
together with three known triterpenoids 2α,3β,21β-trihydroxyursolic acid 28-O-β-D-glucopyranoside
(5), 2α,3α,19α,23-tetrahydroxyoleanolic acid 28-O-β-D-glucopyranoside (6) and 2α,3α,19α,23-tetra-
hydroxyursolic acid 28-O-β-D-glucopyranoside (7). Their structures were elucidated by extensive NMR
1
13
techniques mainly including 1D NMR ( H- and C-NMR), 2D NMR (COSY, NOESY, HSQC, and
HMBC), and ESI-MS.
Compound 1 was obtained as a white amorphous powder, which gave a positive result in the
Liebermann-Burchard test. Acid hydrolysis of compound 1 with 2 mol/L HCl/1,4-dioxane (1:1, v/v)
furnished glucose, identified by TLC by comparison with an authentic sample. The positive optical
2
D
0
rotation (α
+45.1, c 0.03, H O) indicated the D-configuration of glucose, The sugar identity was
2
1
13
further confirmed by the chemical shifts and coupling constants in the H- and C-NMR spectra. The
−
HR-ESI-MS of 1 showed a quasi-molecular ion peak at m/z 665.3901 [M−H] , indicating a molecular
1
13
formula of C36
of 1 in pyridine-d
seven tertiary methyl groups [δ
H
58
O
10 (calcd. for C36
H
57
O
10, 665.3909, Δamu 2.6 ppm). The H- and C-NMR spectra
showed typical signals for an oleanane pentacyclic triterpenoid skeleton including
0.99,1.17, 1.49, 1.66, 1.79, 1.79, 1.83, (each 3H, s)], as well as one
5.61 (1H, br s), two olefinic carbons (δ 124.5 and 144.2) and an ester carbonyl at
4.87 (1H, s), 4.30 (1H,
5
H
olefinic proton at δ
H
C
1
δ
C
177.17. The H-NMR spectra of 1 exhibited four oxymethine protons at δ
H
m), 3.63 (1H, s), and 3.43 (1H, d, J = 9.0 Hz). The data thus suggested that 1 is an oleanane-type
triterpene with four hydroxy groups, a trisubstituted double bond, and a carboxyl. Comparison of the
NMR spectroscopic data of 1 with those of arjunetin [7] demonstrated that the two compounds were
almost identical, except for an additional hydroxyl group at C-6 (δ 68.3). These data suggested that 1
C
is a 6-oxygenated derivative of arjunetin, which was further confirmed by HMBC and NOESY
experiments on 1. The existence of four hydroxy groups at C-2, C-3, C-6 and C-19 was supported by
the HMBC spectrum. HMBC correlations (Figure 2) were observed between H-1 (δ
and C-25 (δ 18.9), C-4 (δ 40.2), C-2 (δ 69.3), C-3 (δ 84.4); between H-2 (δ
3.43) and C-2 (δ 69.3), C-4 (δ 40.2), C-24 (δ 19.0); between H-19 (δ
29.5), C-17 (δ 47.0); between H-5 (δ 1.21), H-7 (δ 2.00) and C-6 (δ
H
2.35 and δ
4.30) and C-3 (δ
3.63) and
68.3). The
H
1.40)
C
C
C
C
H
C
8
4.4); H-3 (δ
H
C
C
C
H
C-21 (δ
C
C
H
H
C
orientations of the hydroxyls at C-2, C-3, C-6 and C-19 were determined using NOESY correlations.

Molecules 2015, 20
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The NOESY correlation of H-3 (δ
H
3.43) with H-23 (δ
H
1.43) indicated that the hydroxyl at C-3 should
be β-oriented; the NOESY correlations of H-2 (δ
H
4.30) with H-24 (δ
H
1.79) and H-25 (δ
4.87) with H-5 (δ
1.49) implied that the 6-OH group had a β-orientation; the NOESY correlations of
H
1.79)
implied that 2-OH group had an α-orientation; the NOESY correlations of H-6 (δ
H
H
1
.21) and H-23 (δ
H
H-19 (δ
H
3.63) with H-30 (δ
H
1.66) implied that the 19-OH group had an α-orientation; Therefore, the
1
aglycon moiety of 1 was identified as 2α,3β,6β,19α-tetrahydroxyoleanolic acid. In the H-NMR
spectrum of 1, the relatively large 3JH-1,H-2 coupling constant of the anomeric proton at δ
D-glucopyranosyl moiety (J = 7.8 Hz) indicated a β-configuration for D-glucose. HMBC correlations
between the anomeric proton at δ 6.36 (1H, d, J = 7.8 Hz) and the carbon signal at C-28 (δ 177.7)
6.36 of the
H
H
C
indicated that a β-D-glucopyranosyl moiety was attached to the C-28 position of the aglycone. On the
basis of the foregoing evidence, the structure of 1 was determined as 2α,3β,6β,19α-tetrahydroxyoleanolic
acid 28-O-β-D-glucopyranoside.
Figure 2. Key HMBC and NOESY correlations of compound 1.
Compound 2 was obtained as a white amorphous powder, which gave a positive result in the
Liebermann-Burchard test. Acid hydrolysis of compound 2 with 2 mol/L HCl/1,4-dioxane (1:1, v/v)
furnished glucose, identified by TLC comparison with an authentic sample. The positive optical rotation
2
D
0
(
α
+46.2, c 0.03, H O) indicated the D-configuration of glucose. In the (−) and (+)-ESI-MS of 2,
2
−
+
quasimolecular ion peaks were observed at m/z: 649 [M−H] and 673 [M+Na] , respectively. The
−
HR-ESI-MS (m/z 649.3962 [M−H] ) analysis revealed the molecular formula of 2 to be C36
H
58
O
10
1
13
(
calcd. for C36
showed typical signals for an oleanane pentacyclic triterpenoid skeleton including seven tertiary
methyl groups [δ 0.88, 0.97, 1.06, 1.11, 1.19, 1.23, 1.54, (each 3H, s)], as well as one olefinic proton
at δ 5.57 (1H, br s), a pair of olefinic carbons at δ 124.3 and 145.3, typical for a double bond at C-12
13) in an oleanane pentacyclic triterpenoid skeleton [6,7] and a carboxyl carbon at δ 181.3. The
1H-NMR spectra of 2 exhibited three oxymethine protons at δ
4.46 (1H, m), 4.05 (1H, d, J = 2.4 Hz), 3.63
1H, s), The data thus suggested that the aglycon moiety of 2 is an oleanane-type triterpene with three
H
57
O
10, 649.3968, Δamu 0.9 ppm). The H- and C-NMR spectra of 2 in pyridine-d
5
H
H
C
(
C
H
(
hydroxy groups, a trisubstituted double bond, and a carboxyl. Comparison of the NMR spectroscopic
data of 2 with those of 2α,3α,19α-dihydroxyoleanolic acid 28-O-β-D-glucopyranoside [7] demonstrated
that the aglycon moiety of the two compounds were almost identical. These data suggesting that 2 has
the same aglycon moiety as 2α,3α,19α-dihydroxyoleanolic acid 28-O-β-D-glucopyranoside were
further confirmed by HMBC and NOESY experiments on 2. The existence of three hydroxy groups at

Molecules 2015, 20
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C-2, C-3 and C-19 was supported by the HMBC spectrum, HMBC correlations (Figure 3) were
observed between H-1 (δ 2.00 and δ 1.86) and C-25 (δ 16.9), C-4 (δ 39.2), C-2 (δ 76.9); between
H-2 (δ 4.46) and C-3 (δ 79.0); between H-3 (δ 4.05) and C-2 (δ 76.9), C-5 (δ 49.5); between H-19
3.63) and C-21 (δ 29.6), C-17 (δ 44.6). The configuration of the hydroxyls at C-2, C-3 and C-19
were determined using NOESY correlations. The NOESY correlation of H-2 (δ 4.46) with H-24 (δ
.88) and H-25 (δ 0.97) indicated that the hydroxyl at C-2 should be in an α-orientation; the NOESY
correlations of H-3 (δ 4.05) with H-24 (δ 0.88) implied that 3-OH group had an α-orientation; the
NOESY correlations of H-19 (δ 3.63) with H-30 (δ 1.54) implied that 19-OH group had an
α-orientation. Therefore, the aglycon moiety of 2 was identified as 2α,3α,19α-trihydroxyoleanolic acid.
H
H
C
C
C
H
C
H
C
C
(
δ
H
C
C
H
H
0
H
H
H
H
H
1
3
In the H-NMR spectrum of 2, the relatively large JH-1,H-2 coupling constant of the anomeric proton at
5.16 of the D-glucopyranosyl moiety (J = 7.8 Hz) indicated a β-configuration for D-Glc. HMBC
correlations between the anomeric proton at δ 6.36 (1H, d, J = 7.8 Hz) and the carbon signal at C-2
76.9) indicated that a β-D-glucopyranosyl moiety was attached to the C-2 position of the aglycone.
δ
H
H
(
δ
C
On the basis of the foregoing evidence, the structure of 2 was determined as 2-O-β-D-
glucopyranosyloxy-3α,19α-dihydroxyoleanolic acid.
Figure 3. Key HMBC and NOESY correlations of compound 2.
Compound 3 was obtained as a white amorphous powder, which gave a positive result in the
Liebermann-Burchard test. Acid hydrolysis of compound 3 with 2 mol/L HCl/1,4-dioxane (1:1, v/v)
furnished glucose, identified by TLC comparison with an authentic sample. The positive optical
2
D
0
rotation (α
+45.7, c 0.03, H O) indicated the D-configuration of glucose. In the (−) and (+)-ESI-MS
2
−
+
of 3, quasimolecular ion peaks observed at m/z: 649 [M−H] and 673 [M+Na] indicated the molecular
weight of 3 is 650. The HR-ESI-MS of 3 showed a quasi-molecular ion peak at m/z 649.3952 [M−H]−,
indicating a molecular formula of C36
1H and 13C-NMR spectra of 3 in pyridine-d
triterpenoid skeleton, including six tertiary methyl groups [δ
H
58
O
10 (calcd. for C36
H
57
O
10, 649.3958, Δamu 0.41 ppm). The
showed typical signals for an ursane pentacyclic
0.86, 0.96, 1.09, 1.22, 1.44, 1.62 (each
5
H
3
H, s)] and one secondary methyl signal at δ
H
1.14 (3H, d, J = 6.6 Hz), as well as one olefinic proton at
1
δ
H
5.56 (1H, br s), two olefinic carbons (δ
C
128.4 and 140.4) and a carboxyl carbon at δ
C
181.1. The H
13
and C-NMR spectra of 3 exhibited two oxymethine protons at δ
H
4.46 (1H, m), 4.03 (1H, d, J = 2.4 Hz)
and one hydroxy group attached to a tertiary carbon. The data thus suggested that 3 is an ursane-type
triterpene with three hydroxy groups, a trisubstituted double bond, and a carboxyl. Comparison of the
NMR spectroscopic data of 3 with those of 2α,3β,19α-trihydroxyurs-12-en-28-O-β-D-glucopyranoside. [7]
demonstrated that the two compounds have the same aglycon moiety, only differing in the orientation
of the hydroxy group at C-3. The existence of three hydroxy groups at C-2, C-3 and C-19 was

Molecules 2015, 20
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supported by the HMBC spectrum, HMBC correlations (Figure 4) were observed between H-1 (δ
H
H
1
.80 and δ
.46) and C-3 (δ
3.05), H-30 (δ
H
1.94) and C-25 (δ
C
17.0), C-4 (δ
4.03) and C-2 (δ
1.44) and C-19 (δ
C
39.0), C-2 (δ
76.6), C-4 (δ
73.1). The configuration of the hydroxyls at C-2,
4.03)
0.86) indicated that the hydroxyl at C-3 should be α-oriented; the NOESY correlations
4.46) with H-24 (δ 0.86) and H-25 (δ 0.96) implied that the 2-OH group had an
α-orientation; the NOESY correlations of H-29 (δ 1.44) with H-18 (δ 3.05) and H-20 (δ 1.50)
implied that 19-OH group had an α-orientation; Therefore, the aglycon moiety of 3 was identified as
C
76.6), C-3 (δ
C
79.1); between H-2 (δ
4
C
79.1); H-3 (δ
H
C
C
39.0), C-24 (δ
C
22.8); between H-18
(δ
H
H
1.14), H-29 (δ
H
C
C-3 and C-19 were determined using NOESY correlations. The NOESY correlation of H-3 (δ
with H-24 (δ
of H-2 (δ
H
H
H
H
H
H
H
H
1
3
2
α,3α,19α-trihydroxyoursolic acid. In the H-NMR spectrum of 3, the relatively large JH-1,H-2 coupling
constant of the anomeric proton at δ 5.14 of D-glucopyranosyl moiety (J = 7.8 Hz) indicated a
β-configuration for D-Glc. HMBC correlations between the anomeric proton at δ 5.14 (1H,d,
J = 7.8 Hz) and the carbon signal at C-2 (δ 76.6) indicated that a β-D-glucopyranosyl moiety was
H
H
C
attached to the C-2 position of the aglycone. On the basis of the foregoing evidence, the structure of 3
was determined as 2-O-β-D-glucopyranosyloxy-3α,19α-dihydroxyursolic acid.
Figure 4. Key HMBC and NOESY correlations of compound 3.
Compound 4 was obtained as a white amorphous powder, which gave a positive result in the
Liebermann-Burchard test. Acid hydrolysis of compound 4 with 2 mol/L HCl/1,4-dioxane (1:1, v/v)
furnished glucose, identified by TLC comparison with an authentic sample. The positive optical
2
D
0
rotation (α
+45.7, c 0.03, H
2
O) indicated the D-configuration of glucose. The HR-ESI-MS of 4
−
showed a quasi-molecular ion peak at m/z 665.3901 [M−H] , indicating a molecular formula of
1
13
C
36
H
58
O
10 (calcd. for C36
H
57
O
10, 665.3909, Δamu 1.2 ppm). The H and C-NMR spectra of 4 in
showed typical signals for an ursane pentacyclic triterpenoid skeleton, including six
tertiary methyl groups [δ 1.04, 1.07, 1.22, 1.25, 1.33, 1.38 (each 3H, s)] and one secondary methyl
signals at δ 0.94 (3H, d, J = 6.6 Hz), as well as one olefinic proton at δ
carbons (δ
exhibited three oxymethine protons at δ
pyridine-d
5
H
H
H
5.36 (1H, br s), two olefinic
1
13
C
129.9 and 139.9) and an ester carbonyl carbon at δ
C
178.5. The H and C-NMR spectra of 4
H
4.38 (1H, s), 4.02 (1H, m), 3.29 (1H, d, J = 2.4 Hz) and
one hydroxy group attached to a tertiary carbon. The data thus suggested that 4 is an ursane-type
triterpene with four hydroxy groups, a trisubstituted double bond, and a carboxyl. Comparison of the
NMR spectroscopic data of 4 with those of 2α,3β,19α-trihydroxyurs-12-en-28-O-β-D-glucopyranoside [7]
demonstrated that two compounds were almost identical, except for an additional hydroxyl group at
C-6 (δ
C
69.2). This was further confirmed by HMBC and NOESY experiments on 4. The existence of
four hydroxy groups at C-2, C-3, C-6 and C-19 was supported by the HMBC spectrum, HMBC

Molecules 2015, 20
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correlations (Figure 5) were observed between H-1 (δ
H
1.54 and δ
4.02) and C-3 (δ
24.4); between H-19 (δ 3.63) and C-21 (δ
69.2). The configuration of the hydroxyls at C-2,
H
1.28) and C-25 (δ
81.5); H-3 (δ
29.5), C-17 (δ
C
18.5), C-4 (δ
3.29) and C-2
47.0);
C
4
0.1), C-2 (δ
67.1), C-4 (δ
between H-5 (δ
C
67.1), C-3 (δ
40.1), C-24 (δ
1.28), H-7 (δ
C
81.5); between H-2 (δ
H
C
H
(
δ
C
C
C
H
C
C
H
H
1.53) and C-6 (δ
C
C-3, C-6 and C-19 were determined using NOESY correlations. The NOESY correlation of H-3 (δ
.29) with H-24 (δ 1.25) indicated that the hydroxyl at C-3 should be in an α-orientation; the NOESY
correlations of H-2 (δ 4.02) with H-24 (δ 1.25) and H-25 (δ 1.38) implied that the 2-OH group had
an α-orientation; the NOESY correlations of H-6 (δ 4.38) with H-5 (δ 1.28) and H-23 (δ 1.07)
implied that the 6-OH group had a β-orientation; the NOESY correlations of H-19 (δ 3.63) with H-30
1.66) the NOESY correlations of H-29 (δ 1.22) with H-18 (δ 2.54) and H-20 (δ 1.36) implied
that the 19-OH group had an α-orientation. Therefore, the aglycon moiety of 4 was identified as
H
3
H
H
H
H
H
H
H
H
(
δ
H
H
H
H
1
3
2
α,3α,6β,19α-tetrahydroxyursolic acid. In the H-NMR spectrum of 4, the relatively large JH-1,H-2
coupling constant of the anomeric proton at δ 5.32 of the D-glucopyranosyl moiety (J = 8.4 Hz)
indicated a β-configuration for D-Glc. HMBC correlations between the anomeric proton at δ 5.32
1H, d, J = 8.4 Hz) and the carbon signal at C-28 (δ 178.5) indicated that a β-D-glucopyranosyl
H
H
(
C
moiety was attached to the C-28 position of the aglycone. On the basis of the foregoing evidence, the
structure of 4 was determined as 2α,3α,6β,19α-tetrahydroxyursolic acid 28-O-β-D-glucopyranoside.
Figure 5. Key HMBC and NOESY correlations of compound 4.
The structures of the three known triterpenoids 2α,3β,21β-trihydroxy ursolic acid 28-O-β-D-
glucopyranoside (5) [8], 2α,3α,19α,23-tetrahydroxyoleanolic acid 28-O-β-D-glucopyranoside (6) [9]
and 2α,3α,19α,23-tetrahydroxyursolic acid 28-O-β-D-glucopyranoside (7) [10] were determined by
comparison of their NMR spectral data with those reported in the literature.
3. Experimental Section
3
.1. General Experimental Procedures
Optical rotations were measured on an Autopol IV-T/V (Rudolph Research Analytical,
Hackettstown, NJ, USA). UV spectra were recorded in MeOH on a Jasco V650 spectrophotometer
1
13
(
JASCO, Inc., Easton, MD, USA). The H (600 MHz), C- (150 MHz), and 2D-NMR spectra were
recorded on a Bruker AVANCE III 600 instrument using TMS (tetramethylsilane) as an internal
reference (Bruker Company, Billerica, MA, USA). HRTOFMS data were obtained on an Agilent
7
890–7000 A mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Preparative HPLC

Molecules 2015, 20
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(
high-performance liquid chromatography) was conducted with an Agilent Technologies 1200 series
instrument with an multiple wavelength detector using a YMC-pack ODS (Octadecylsilyl)-A column
5 µm, 250 × 20 mm). Column chromatography was performed with silica gel (200–300 mesh,
(
Qingdao Marine Chemical Ltd., Qingdao, China), Develosil ODS (50 µm, Nomura Chemical Co. Ltd.,
Osaka, Japan), and Sephadex LH-20 GE Healthcare Bio-Sciences AB, Uppsala, Sweden). TLC (thin
layer chromatography) was carried out with glass precoated with silica gel GF254. Spots were
visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating. All
reagents and solvents are analystical grade.
3
.2. Plant Material
The aerial parts of C. kwangtungensis Chun were collected from Pingxiang, Jiangxi province of
China, in July 2012. The plant was identified by Guiping Yuan at Jiangxi Provincial Institute for Drug
and Food Control, China. A voucher specimen (No. 20120715) is deposited in the Herbarium of
Jiangxi Provincial Institute for Drug and Food Control.
3.3. Extraction and Isolation
The aerial parts of C. kwangtungensis (10.5 kg) were extracted three times with 95% EtOH under
reflux (2 h each). The extracted solution was evaporated under reduced pressure to yield a dark-brown
residue (1.2 kg). The residue was suspended in water (20 L) and then successively partitioned with
petroleum ether (3 × 20 L), EtOAc (3 × 20 L), and n-BuOH (3 × 20 L). After removing the solvent, the
EtOAc-soluble portion (130 g) was fractionated via silica gel column chromatography (CC), eluting
with CHCl
Fraction A2 (7.8 g) was subjected to silica gel CC and eluted with CHCl
to afford nine fractions (A2-1-A2-9). Fraction A2-4 (2.5 g) was separated by ODS CC (50 µm,
0%–100%, MeOH/H O) to give four subfractions (A2-4-1-A2-4-4). Subfraction A2-4-3 (1.1 g) was
3
/MeOH (5:1, v/v), to give 10 major fractions A1-A10 on the basis of TLC analysis.
3
/MeOH (30:1–1:1, v/v)
2
2
separated by Sephadex LH-20 CC using MeOH to afford five fractions (A2-4-3-1–A2-4-3-5) on
the basis of TLC analysis. Fraction A2-4-3-3 (108 mg) was further separated by preparative HPLC
(
YMC-ODS-A, 5 µm, 250 mm × 20 mm, detection at 210 nm) using 23% CH
mobile phase to yield 1 (20.2 mg) and 2 (4.0 mg). A2-4-2 (1.1 g) was subjected to silica gel CC
and eluted with CHCl /MeOH (12:1–4:1) to afford three fractions (A2-4-2-1-A2-4-2-3). Subfraction
A2-4-2-2 (108 mg) was separated by preparative HPLC (YMC-ODS-A, 5 µm, 250 mm × 20 mm,
detection at 210 nm) using 23% CH CN–H O (7 mL/min) to yield 3 (4.5 mg), 4 (21.3 mg) and 5
5.8 mg). Fraction 5 (8.9 g) was subjected to silica gel CC and eluted with CHCl /MeOH (30:1–1:1,
v/v) to afford five fractions (A5-1-A5-5). A5-4 (2.7 g) was subjected to ODS CC (50 µm, 20%–100%,
MeOH-H O) to afford four subfractions (A5-4-1-A5-4-4). A5-4-3 (1.31 g) was subjected to silica gel
CC and eluted with CHCl /MeOH (30:1–1:1, v/v) to afford three fractions (A5-4-3-1~A5-4-3-3),
A5-4-3-1 (217 mg) separated by preparative HPLC (YMC-ODS-A, 5 µm, 250 mm × 20 mm, detection
at 210 nm) using 23% CH CN–H O (7 mL/min) to yield 6 (5.3 mg)and 7 (16.8 mg).
3
CN–H
2
O (7 mL/min) as
3
3
2
(
3
2
3
3
2

Molecules 2015, 20
.4. Acid Hydrolysis of Compounds 1–4
Compounds 1 (1.0 mg), 2 (1.0 mg), 3 (1.0 mg) and 4 (1.0 mg) were heated in an ampule with
9079
3
aqueous 2 mol/L HCl/1,4-dioxane (1:1, 2 mL) at 80 °C for 6 h. The aglycone was extracted with
chloroform (3 × 3 mL). The aqueous layer was evaporated under reduced pressure and subjected to the
column chromatography over Sephadex LH-20, eluting with CH
residue. Compound 1, 2, 3 and 4 gave D-glucose which was identified by TLC comparison with a
standard sample (CH CN/H O (6:1); R = 0.35 and its positive optical rotation.
3
CN/H
2
O (8:1) to yield the sugar
3
2
f
3
.5. The Physicochemical Data of Compounds 1–7
α,3β,6β,19α-Tetrahydroxyoleanolic Acid 28-O-β-D-Glucopyranoside (1). White amorphous powder;
2
2
0
1

α
D
–12.5 (c 0.12, MeOH); UV (MeOH) λmax (logε): 207.6 (3.33) nm; for H-NMR (600 MHz, C
5
D
5
N)
N) spectral data, see Table 1; HR-ESI-MS m/z 665.3901 [M−H]−,
H O10, 665.3909, Δamu 2.6 ppm).
1
3
and C-NMR (150 MHz, C
calcd for C36
5
D
5
(
57
2
D
0
2
-O-β-D-Glucopyranosyloxy-3α,19α-dihydroxyoleanolic Acid (2). White amorphous powder; α
1
−
23.3 (c 0.03, MeOH); UV (MeOH) λmax (logε): 206.2 (3.22) nm; for H-NMR (600 MHz, C
5
D
5
N) and
13C-NMR (150 MHz, C
D
5
N) spectral data, see Table 1; HR-ESI-MS m/z 649.3962 [M−H] (calcd for
−
5
C
36
H
57
O10, 649.3968, Δamu 0.9 ppm).
1
13
Table 1. H-NMR (600 MHz) and C-NMR (150 MHz) spectral data of 1–4 (δ in ppm,
J in Hz, in pyridine-d ).
5
1
2
3
4
No.
C
H
C
H
C
H
C
H
1
.40, 1H, br t,
1.86, 1H, br t,
J = 12.0 Hz
2.00, 1H, m
4.46, 1H, m
1.80, 1H, br t,
J = 12.0 Hz
1.94, 1H, m
4.46, 1H, m
1
50.4
39.8
39.9
44.9
1.28, 1H, s
J = 12.0 Hz
.35, 1H, m
69.3 4.30, 1H, m 76.9
2
1.54, 1H, m
4.02, 1H, m
2
3
76.6
67.1
3
.43, 1H, d,
84.4
79 4.05,1H, d, J = 2.4 Hz 79.1 4.03, 1H, d, J = 2.4 Hz 81.5 3.29, 1H, d, J = 2.4 Hz
J = 9.0 Hz
4
5
6
40.2
39.2
39.0
48.8
19.0
40.1
49.7
69.2
57.4 1.21, 1H, s 49.5
68.3 4.87, 1H, s 19.1
1.68, 1H, m
1.34, 1H, m
1.66, 1H, m
1.33, 1H, m
1.47 m
1.28, 1H, s
4.38, 1H, s
1
.49 m
1.36, 1H, m
.58, 1H, m
7
41.3 2.00, 2H, m 34.1
33.9
1.35, 1H, m
1.65, 1H, m
40.6
1.53, 2H, m
1.90, 1H, m
1
8
9
42.1
49.5 2.14, 1H, m 48.7
39.0 39.2
24.8 2.33, 1H, m 24.8
40.7
41.0
48.1
41.7
49.5
2.08, 1H, m
2.03, 1H, m
1
1
1
1
0
1
39.0
38.3
2.01, 1H, m
5.57, 1H, s
24.6
2.00, 1H, s
5.56, 1H, s
24.4
2.09, 2H, m
5.36, 1H, s
2 124.5 5.61, 1H, s 124.3
3 144.2 145.3
128.4
140.4
129.9
139.9

Molecules 2015, 20
9080
Table 1. Cont.
1
2
3
4
No.
C
H
C
H
C
H
C
H
1
1
4
5
43.3
42.7
42.7
29.7
43.2
29.3
29.6 1.32, 1H, m 29.7
.08, 1H, m
28.7 2.17, 1H, m 28.9
.83, 1H, m
1.33, 1H, m
2.12, 1H, m
2.16, 1H, m
2.80, 1H, m
1.25, 1H, m
2.33, 1H, m
1.33, 1H, m
2.08, 1H, m
1.03, 1H, m
1.91, 1H, m
1.26, 1H, m
1.77, 1H, m
2
1
6
27.4
27.1
2
1
1
1
2
2
7
8
9
0
1
47.0
46.6
49.3
55.0
73.2
42.8
26.9
49.6
55.0
73.7
43.0
26.6
45.1 3.58, 1H, s 45.3
81.6 3.63, 1H, s 81.8
3.60, 1H, m
3.63, 1H, t
3.05, 1H, t
2.54, 1H, s
36.0
29.5 1.07, 1H, m 29.6
.48, 1H, t
36.2
1.50, 1H, m
2.04, 1H, m
1.36, 1H, m
1.64, 1H, m
1.25, 1H, m
2
1
.35, 1H, m
3.11, 1H, m
2.62, 1H, m
J = 6.0 Hz
2
2
33.4 2.00, 1H, m 33.8
2.04, 1H, m
2.17, 1H, m
1.23, 3H, s
0.88, 3H, s
0.97, 3H, s
1.06, 3H, s
1.54, 3H, s
39.2
2.08, 1H, m
2.16, 1H, m
1.22, 3H, s
0.86, 3H, s
0.96, 3H, s
1.09, 3H, s
1.62, 3H, s
38.8
1.66, 1H, m
1.79, 1H, m
1.07, 3H, s
1.25, 3H, s
1.38, 3H, s
1.04, 3H, s
1.33, 3H, s
2
.08, 1H, m
2
2
2
2
2
2
2
3
3
4
5
6
7
29.6 1.49, 3H, s 29.9
19.0 1.79, 3H, s 22.8
18.9 1.79, 3H, s 17.0
19.8 1.83, 3H, s 18.1
25.3 0.99, 3H, s 25.3
29.8
22.8
17.0
17.7
25.1
181.1
27.5
29.6
24.4
18.5
18.7
24.7
178.5
27.2
8 177.7
181.3
9
0
29.2 1.17, 3H, s 29.3
25.3 1.66, 3H, s 25.3
1.19, 3H, s
1.54, 3H, s
1.44, 3H, s
1.22, 3H, s
17.3 1.14, 3H, d, J = 6.6 Hz 16.6 0.94, 3H, d, J = 6.6 Hz
Glc
6
.36, 1H, d,
1
96.4
74.6
79.7
71.7
79.3
62.6
104.2 5.16, 1H, d, J = 7.2 Hz 104.1 5.14, 1H, d, J = 7.8 Hz 95.9 5.32, 1H, d, J = 7.8 Hz
75.8 4.07,1H, t, J = 7.2 Hz 75.9 4.07, 1H, t, J = 7.8 Hz 73.9 3.35, 1H, t, J = 7.8 Hz
78.3 4.28,1H, d, J = 9.0 Hz 78.3 4.29, 1H, d, J = 8.4 Hz 78.3 3.35, 1H, d, J = 8.4 Hz
72.3 4.30,1H, t, J = 9.0 Hz 72.2 4.31, 1H, t, J = 8.4 Hz 71.3 3.40, 1H, t, J = 8.4 Hz
J = 7.8 Hz
4
.24, 1H, t,
2
3
4
5
6
J = 7.8 Hz
4
.02, 1H, d,
J = 9.0 Hz
4
.39, 1H, t,
J = 9.0 Hz
4
.30, 1H, t,
4.03, 1H, dd, J = 8.4 Hz
78.8 4.32, 1H, t, J = 9.0 Hz 78.9 4.33, 1H, t, J = 8.4 Hz 78.6
J = 4.2 Hz
J = 9.0 Hz
4
.41, 1H, t,
4.37, 1H, dd, J = 4.8 Hz
J = 12.0 Hz
4.37, 1H, dd, J = 8.4 Hz
J = 11.4 Hz
3.70, 1H, dd, J = 4.2 Hz
J = 12.0 Hz
63.3
63.2
62.4
J = 9.6 Hz
4
.54, 1H, dd, J = 2.4 Hz
J = 12.0 Hz
4.54, 1H, dd, J = 2.4 Hz
J = 11.4 Hz
3.81, 1H, dd, J = 1.8 Hz
J = 12.0 Hz
4
.44, 1H, m
2
D
0
2
-O-β-D-Glucopyranosyloxy-3α,19α-dihydroxyursolic Acid (3). White amorphous powder; α
−13.0
1
(
c 0.1, MeOH); UV (MeOH) λmax (logε): 208.6 (3.16) nm; for H-NMR (600 MHz, C
13C-NMR (150 MHz, C
5
D
5
N) and
−
5
D N) spectral data, see Table 1; HR-ESI-MS m/z 649.3952 [M−H] (calcd for
5
C
36
57
H O10, 649.3958, Δamu 0.41 ppm).

Molecules 2015, 20
9081
2
α,3α,6β,19α-Tetrahydroxyursolic Acid 28-O-β-D-Glucopyranoside (4). White amorphous powder;
20
1
α
D
−15.0 (c 0.08, MeOH); UV (MeOH) λmax (logε): 210.1 (3.55) nm; for H-NMR (600 MHz, C
5
D
5
N)
N) spectral data, see Table 1; HR-ESI-MS m/z 665.3901 [M–H] (calcd
H O10, 665.3909, Δamu 1.2 ppm).
1
3
−
and C-NMR (150 MHz, C
5
D
5
for C36
57
2
α,3β,21β-Trihydroxyursolic acid 28-O-β-D-glucopyranoside (5), 2α,3α,19α,23-tetrahydroxyoleanolic
acid 28-O-β-D-glucopyranoside (6), and 2α,3α,19α,23-tetrahydroxyursolic acid 28-O-β-D-glucopyranoside
1
13
(
7), for H-NMR (600 MHz, C
5
D
5
N) and C-NMR (150 MHz, C
5
5
D N) spectral data, see Table 2.
1
13
Table 2. H-NMR (600 MHz) and C-NMR (150 MHz) spectral data of 5–7 (δ in ppm, J
in Hz, in pyridine-d ).
5
Position
5
6
7
1
46.7
0.95 (1H, m)
42.7
1.90 (1H, m)
2.00 (1H, m)
4.08 (1H, m)
3.78 (1H, d, J = 2.4 Hz)
48
1.23 (1H, m)
2.08 (1H, m)
4.10 (1H, m)
3.76 (1H, d, J = 3.6 Hz)
2
.25 (1H, m)
2
3
4
5
67.2
83.1
39
3.31 (1H, m)
66.7
79.9
42.6
44
69.4
78.8
44.2
48.5
2.95 (1H, d, J = 9.6 Hz)
55.6
0.93 (1H, m)
1.41 (1H, s)
1.21 (1H, m)
1.34 (1H, m)
1.25 (1H, m)
1.40 (1H, m)
1.33 (1H, m)
1.48 (2H, m)
1.70 (2H, m)
2.03 (1H, m)
1
.23 (1H, m)
1.38 (1H, m)
.25 (1H, m)
1
9
6
18.5
19.3
1
3
3.7
7
8
9
32.8
39
1.42(1H, m)
1.93 (1H, m)
2.16 (1H, m)
33.9
40.8
49
41.2
48.3
38.2
25
47.8
37.7
23.3
2.06 (1H, m)
1
1
1
1
1
1
0
1
2
3
4
5
39.1
24.8
1283.6
144.8
42.7
29.7
2.00 (1H, m)
2.01 (1H, m)
122.3 4.76 (1H, d, J = 4.5 Hz) 128.7
5.56 (1H, br s)
5.54 (1H, br s)
143.7
41.4
28.5
139.6
42.7
29.7
0.98 (1H, m)
.83 (1H, m)
1.06 (1H, m)
.68 (1H, m)
1.34 (1H, m)
2.10 (1H, m)
2.14 (1H, m)
2.62 (1H, m)
1.31 (1H, m)
2.04 (1H, m)
2.08 (1H, m)
2.74 (1H, m)
1
1
6
24.3
27.1
26.7
1
1
1
1
7
8
9
47
49.1
54.9
73.1
47
41.8
46.6
2.52 (1H, s)
1.06 (1H, m)
2.52 (1H, s)
45.1
81.5
3.52 (1H, s)
3.57 (1H, s)
2
.16 (1H, m)
2
2
0
1
36
42.1
26.6
1.43 (1H, m)
2.00 (1H, m)
36
71.4
3.53 (1H, m)
1.91 (1H, m)
29.5
1.88 (2H, m)
3
.13 (1H, m)
2
2
2
3
41.5
29.5
38.9
71
2.04 (1H, m)
2.14 (1H, m)
33.5
67
2.04 (1H, m)
2.10 (1H, m)
2
.22 (1H, m)
0.84 (3H, s)
3.75 (1H, d, 10.8 Hz)
3.57 (1H, d, 10.2 Hz)
3.73 (1H, d, 10.2 Hz)
3
.92 (1H, d, 10.8 Hz)

Molecules 2015, 20
9082
Table 2. Cont.
Position
5
6
7
2
2
2
2
2
2
3
4
5
6
7
8
9
0
17.3
16.7
17
1.06 (3H, s)
1.00 (3H, s)
0.91 (3H, s)
1.41 (3H, s)
18.2
17.6
18
0.90 (3H, s)
14.7
17,8
18.2
25.9
177.8
29.4
0.99 (3H, s)
1.22 (3H, s)
1.10 (3H, s)
1.56 (3H, s)
1.09 (3H, s)
1.26 (3H, s)
1.65 (3H, s, )
25.8
176.6
29.7
20.1
27.5
177.9
24.7
1.12 (3H, s)
1.09 (3H, s)
1.39 (3H, s)
1.15 (3H, s)
1.17 (3H, s)
17.2 1.07 (3H, d, J = 6.6 Hz) 25.1
Glc
1
2
3
4
5
6
′
′
′
′
′
′
96.6
74
5.44 (1H, d, J = 7.8 Hz) 96.4 6.30 (1H, d, J = 7.8 Hz) 96.4 6.30 (1H, d, J = 7.8 Hz)
3.33 (1H, m)
3.43 (1H, m)
3.3 (1H, m)
3.31 (1H, m)
3.64 (1H, m)
74.6
79.8
71.7
79.5
62.7
4.22 (1H, m)
4.02 (1H, m)
4.33 (1H, m)
4.27 (1H, m)
4.35 (1H, m)
4.37 (1H, m)
74.6
79.9
71.6
79.8
62.7
4.21 (1H, m)
3.98 (1H, m)
4.32 (1H, m)
4.25 (1H, m)
4.37 (1H, m)
4.40 (1H, m)
78.9
71.3
79.4
61.9
3
.85 (1H, m)
4
. Conclusions
Four new triterpenoids which were identifed as 2α,3β,6β,19α-tetrahydroxyoleanolic acid
8-O-β-D-glucopyranoside (1), 2-O-β-D-glucopyranosyloxy-3α,19α-dihydroxyoleanolic acid (2),
-O-β-D-glucopyranosyloxy-3α,19α-dihydroxyursolic acid (3) and 2α,3α,6β,19α-tetrahydroxyursolic acid
8-O-β-D-glucopyranoside (4), were isolated together with three known triterpenoids identified as
α,3β,21β-trihydroxyursolic acid 28-O-β-D-glucopyranoside (5), 2α,3α,19α,23-tetrahydroxyoleanolic
2
2
2
2
acid 28-O-β-D-glucopyranoside (6), and 2α,3α,19α,23-tetrahydroxyursolic acid 28-O-β-D-glucopyranoside
7) from the aerial parts of Callicarpa kwangtungensis. This finding represents an addition to the
(
ongoing research on the pharmacological activity of this plant, which may be helpful to understand the
use of Callicarpa kwangtungensis in traditional medicine and should continue to clarify its actual
health benefits.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/05/9071/s1.
Acknowledgments
This study was supported financially by the National Natural Science Foundation of China (NSFC,
Grant No. 81460589) and Natural Science Foundation of Jiangxi, China (No. 20114BAB205070 and
No. 20122BAB215062). The authors thank A.H.L. at the Center of Analysis and Testing Nanchang
University for NMR measurements.

Molecules 2015, 20
9083
Author Contributions
Tengfei Ji conceived and designed the experiments; Guo-Ping Zhou, Yan Yu performed the
experiments and analyzed the data; Ming-Ming Yuan, Tengfei Ji and Hui-Zheng Fu contributed
materials and analysis tools; Guo-Ping Zhou, Tengfei Ji and Rui-Jian Zhong wrote the paper.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–7 are available from the authors.
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