New 30-noroleanane triterpenoid saponins from Holboellia coriacea Diels

Article abstract of DOI:10.3390/molecules21060734

Three new 30-noroleanane triterpenoid saponins, akebonoic acid 28-O-β-D-glucopyranosyl-(1″→6′)-β-D-glucopyranosyl ester (1), akebonoic acid 28-O-(6″-O-caffeoyl)-β-D-glucopyranosyl-(1″→6′)-β-D-glucopyranosyl ester (Holboelliside A, 2) and 3β,20α,24-trihydroxy-29-norolean-12-en-28-oic acid 3-O-(6′-O-caffeoyl)-β-D-glucopyranoside (Holboelliside B, 3) were isolated from the stems of Holboellia coriacea Diels, together with five known compounds, eupteleasaponin VIII (4), 3α-akebonoic acid (5), quinatic acid (6), 3β-hydroxy-30-norhederagenin (7) and quinatoside A (8). The structures of these compounds were determined on the basis of spectral and chemical evidence. Compounds 1-5 were evaluated for their inhibitory activity against three human tumors HepG2, HCT116 and SGC-7901 cell lines in vitro.

Full text of DOI:10.3390/molecules21060734

molecules
Article
New 30-Noroleanane Triterpenoid Saponins from
Holboellia coriacea Diels
Wenbing Ding 1,2, Ye Li 1, Guanhua Li 1, Hualiang He 1, Zhiwen Li 1 and Youzhi Li 1,2,
*
1
Hunan Provincial Engineering & Technology Research Center for Biopesticide and Formulation Processing,
Hunan Agricultural University, Changsha 410128, China; dingwenb119@hunau.edu.cn (W.D.);
15273361055@139.com (Y.L.); lgh202@126.com (G.L.); hhl_1234@126.com (H.H.);
lizhw809718@aliyun.com (Z.L.)
Hunan Co-Innovation Center for Utilization of Botanical Functional Ingredients, Changsha 410128, China
Correspondence: liyouzhi@hunau.edu.cn; Tel.: +86-731-8461-8163
2
*
Academic Editor: Derek J. McPhee
Received: 10 May 2016; Accepted: 2 June 2016; Published: 4 June 2016
Abstract:
28-O- -D-glucopyranosyl-(1
-D-glucopyranosyl-(1
29-norolean-12-en-28-oic acid 3-O-(61-O-caffeoyl)-
isolated from the stems of Holboellia coriacea Diels, together with five known compounds,
eupteleasaponin VIII ( ), 3 -akebonoic acid ( ), quinatic acid ( ), 3 -hydroxy-30-norhederagenin (
and quinatoside A ( ). The structures of these compounds were determined on the basis of spectral
and chemical evidence. Compounds were evaluated for their inhibitory activity against three
Three new 30-noroleanane triterpenoid saponins,
akeb1o1noic acid
11
1
β
β
Ñ
6 )-
-D-glucopyranosyl ester (
1
), akebonoic acid 28-O-(6 -O-caffeoyl)-
11
1
β
Ñ
6 )-
β
-D-glucopyranosyl ester (Holboelliside A,
2) and 3
β
,20
α
,24-trihydroxy-
) were
β
-D-glucopyranoside (Holboelliside B,
3
4
α
5
6
β
7)
8
1–5
human tumors HepG2, HCT116 and SGC-7901 cell lines in vitro.
Keywords: Holboellia coriacea; nortriterpenoids; saponins; cytotoxicity
1. Introduction
Holboellia coriacea Diels belonging to family Lardizabalaceae, is an evergreen woody vines mainly
distributed in Qinling Mountain region of China at altitudes of 500–2000 m [
coriacea, edible berries, can be used to wine, while the stems and roots of the plant have been used
as Chinese folk medicine for treating arthritis and rheumatism paralysis [ ]. To date there are very
1]. The fruit of H.
1,2
few reports about the chemical constituents of this species. Recently, a phytochemical study revealed
six triterpenoid saponins, including two noroleanane-type ones from a methanol extract of root of
H. coriacea [
Lardizabalaceae, some of which were revealed to show significant bioactivities [
in the chemistry of noroleanane triterpenoids prompted us to continue the phytochemical study of
H. coriacea, whereby three new 30-noroleanane triterpenoid saponins ( ) and five known ones (4–8)
2]. Noroleanane triterpenoids are recognized as characteristic constituents of the family
3–5
]. Our interest
1–
3
were obtained from the stem of the plant. Herein, we report the isolation, structure elucidation, and
cytotoxic activity of these compounds.
2. Results and Discussion
The dried stems of H. coriacea were extracted using 95% aq. EtOH. The EtOH extract residue
was suspended in water and then partitioned successively with petroleum ether and EtOAc. Column
chromatography of the EtOAc-soluble fraction yielded three new triterpenoid saponins (
were identified by NMR techniques and HRESIMS, and five known compounds that were identified
as by comparison of their NMR and MS data with those reported in the literature as eupteleasaponin
1–3), which
4–8
Molecules 2016, 21, 734; doi:10.3390/molecules21060734
www.mdpi.com/journal/molecules

Molecules 2016, 21, 734
2 of 8
VIII (4) [6], 3α
-akebonoic acid (
5
) [7], quinatic acid (
6) [8], 3
β
-hydroxy-30-norhederagenin (
7) [9
] and
quinatoside A (8) [10] (Figure 1).
Figure 1. Structures of compounds 1–8 isolated from H. coriacea, as well as structures of 1a and 3a
mentioned in the text.
2.1. Identification of New Compounds
Compound
1 was obtained as white solid powder (MeOH). The positive HR-ESIMS of 1
showed an [M + Na]+ at m/z 787.4248, which, taken together with the 13C-NMR data analysis,
indicated the molecular formula C41H64O13. The 13C-NMR spectrum displayed 41 signals, of which
12 carbons were assignable to sugar moieties and 29 carbons to the aglycone moiety (Table 1).
On the basis of 13C-NMR and DEPT spectra, the carbons for the aglycone were identified as five
methyls, four olefinic carbons, an oxy-methine carbon, a carbonyl carbon, 10 methylenes, three
methines, and five quaternary carbons. The HSQC spectrum of
1 displayed corresponding five
angular methyl groups (δH 0.94 (s, 3H); 1.02 (s, 3H); 1.12 (s, 3H); 1.20 (s, 3H); 1.22 (s, 3H)), a broad
singlet olefinic proton at δH 5.45, and two exo-methylene protons at δH 4.65 (s) and 4.71 (s). These
NMR data suggested that the structure of
comparison of the 1H and 13C-NMR data with those of closely related analogues, the aglycone was
characterized as 3 -hydroxy-30-norolean-12,20(29)-dien-28-oic acid (akebonoic acid) [ ], a common
triterpene aglycon occurring in the genera of Lardizabalaceae. The -orientation of the hydroxyl
group at C-3 was confirmed by an NOE correlation of H-3 with H
1 should be a noroleanane triterpenoid saponin [4]. After
β
7
β
α
-5 (Figure S7) and the proton
spin-coupling constant of H-3 (δH 3.44 (dd, J = 9.9, 5.6 Hz)). In addition, the H and 13C-NMR
spectra exhibited two anomeric proton signals at δH 6.21 (d, J = 8.0 Hz) and 5.00 (d, J = 7.7 Hz), as
well as twelve carbon signals at δC 95.2 (glc-11), 73.3 (glc-21), 78.1 (glc-31), 70.4 (glc-41), 77.3 (glc-51),
69.0 (glc-61), 104.8 (glc-111), 74.6 (glc-211), 77.8 (glc-311), 71.0 (glc-411), 77.9 (glc-511), and 62.1 (glc-611),
which correspond with that of published data for a sugar moiety of
-D-glucopyranosyl [11]. Furthermore, the sugar moiety was found to be attached to C-28 via an
1
β
-D-glucopyranosyl (111
Ñ
61)
β
1
ester linkage from correlations between H-1 (δH 6.21) and carboxyl carborn (δC 175.3) observed in the
HMBC spectrum (Figure S6). Based upon all o1f1the1above evidence, the structure of
1 was elucidated
as akebonoic acid 28-O-β-D-glucopyranosyl-(1 Ñ6 )-β-D-glucopyranosyl ester.

Molecules 2016, 21, 734
3 of 8
Table 1. NMR spectroscopic data for compound 1 in pyridine-d5.
No.
δc
δ
(mult., J in Hz)
No.
δc
δ (mult., J in Hz)
H
H
Ag-1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
38.5 CH2
27.5 CH2
77.6 CH
38.8 qC
55.3 CH
18.3 CH2
32.6 CH2
39.4 qC
47.6 CH
36.8 qC
23.2 CH2
122.9 CH
143.0 qC
41.5 qC
27.7 CH2
23.0 CH2
46.8 qC
47.0 CH
41.2 CH2
147.9 qC
29.5 CH2
37.1 CH2
0.98 (m); 1.52 (m)
1.79–1.82 (m)
3.44 (dd, 9.9, 5.6)
23
24
25
26
27
28
29
28.2 CH3
16.0 CH3
15.1 CH3
17.0 CH3
25.5 CH3
175.3 qC
106.7 CH2
1.22 (s)
1.02 (s)
0.94 (s)
1.12 (s)
1.20 (s)
0.84 (d, 11.2)
1.36 (m); 1.51 (m)
1.35 (m); 1.47 (m)
4.65 (s); 4.71 (s)
1.63 (dd, 9.7, 7.4)
Glc-11
95.2 CH
73.3 CH
78.1 CH
70.4 CH
77.3 CH
69.0 CH2
6.21 (d, 8.0)
4.12 (m)
4.19 (m)
4.33 (m)
4.07 (m)
21
31
41
51
61
1.90 (m)
5.45 (br s)
4.35 (dd, 12.1, 4.9)
4.69 (br d, 12.1)
5.00 (d, 7.7)
4.00 (t, 7.7)
4.21 (m)
4.19 (m)
3.88 (m)
4.48 (br d, 10.5)
4.32 (m)
1.18 (m); 2.34 (m)
2.06 (m); 2.14 (m)
Glc-111
211
104.8 CH
74.6 CH
77.8 CH
71.0 CH
77.9 CH
62.1 CH2
3.13 (dd, 4.7, 13.4)
2.20 (m); 2.59 (t, 13.4)
311
411
511
2.09 (m); 2.20 (m)
1.74 (m); 2.03 (m)
611
δ in ppm; 1H-NMR: 400 MHz and 13C-NMR 100 MHz.
Compound
2
gave quasi-molecular ions at m/z 949 [M + Na]+ and 961 [M + Cl]´ by ESI-MS.
1
The molecule formula of C50H70O16 was confirmed by HR-ESIMS. By comparison of the H and
13C-NMR data (Table 2) with those of
acid) and sugar moiety as those in
1
, compound
2 was found to have the same aglycone (akebonoic
1, but an additional trans-caffeoyl moiety which was proposed
according to the presence of an aromatic ABX system (δH 7.54, br s; 7.09, dd, J = 8.2, 1.9 Hz; 7.17, d,
J = 8.2 Hz), together with two coupled doublets (J = 15.8 Hz) at δH 6.61 and 7.91 and the signal of an
ester carbon C=O at δC 167.1 [12]. The trans-caffeoyl moiety was deduced to be bound at C-611 of the
sugar moiety via an ester linkage from the deshielded signal of the protons H-611 (Table 2) and from
11
long-range correlations of H-6 (4.88 (dd, J = 11.8, 5.5 Hz)/5.03 (dd, J = 11.8, 1.6 Hz)) with C=O (δC 167.1)
observed in the HMBC spectrum (Figure S13). The aforementioned structure was confirmed by alkaline
and acid hydrolysis of
determined as akebonoic acid 28-O-(6 -O-caffeoyl)-
ester, a new caffeoylation of nortriterpenoid saponin that we have named Holboelliside A.
Compound was obtained as a light yellow powder. The molecular formula was shown to be
C44H62O13 by its HR-ESIMS and 13C-NMR data. Its 1H- and 13C-NMR spectra (Table 3) were similar
to those of compound , eupteleasaponin VIII [ ], except for the presence of a typical ABX aromatic
2
, which yielded akebonoic acid (1a) and D-glucose. Therefore, compound
2
was
11
11
1
β
β
-D-glucopyranosyl-(1
Ñ6 )-
-D-glucopyranosyl
3
4
6
spin system at δH 7.49 (1H, d, 1.6 Hz), 7.18 (1H, d, 8.2 Hz), and 7.05 (1H, dd, 8.2, 1.6 Hz), as well as
two coupled doublets (J = 15.8 Hz) protons for a trans-caffeoyl unit [12]. The deshielded signal of
the protons H-61 (4.95 (dd, J = 11.4, 4.7 Hz)/5.03 (br d, J = 10.1 Hz)) of the glucose moiety and the
1
correlation between H-6 and C=O (δC 167.4) in the HMBC spectrum indicated that the trans-caffeoyl
moiety is attached to C-61 via an ester linkage. Moreover, In the NOESY spectrum (Figure S21),
H
α
-5 was correlated with H-3 and H3-23, and H
stereostructures of the aglycone should be 3 ,20 ,24-trihydroxy-29-norolean-12-en-28-oic acid [13].
Consequently, the structure of was determined to be 3 ,20 ,24-trihydroxy-29-norolean-12-en-28-oic
-D-glucopyranoside, a cafeoyl ester of that we have named Holboelliside B.
β-18 was correlated with H3-29, confirming the
β
α
3
β
α
1
acid 3-O-(6 -O-caffeoyl)-
β
4

Molecules 2016, 21, 734
4 of 8
Table 2. NMR spectroscopic data for compound 2 in pyridine-d5.
No.
δc
δ
(mult., J in Hz)
No.
δc
δ (mult., J in Hz)
H
H
Ag-1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
38.4 CH2
27.5 CH2
77.6 CH
38.8 qC
55.3 CH
18.2 CH2
32.6 CH2
39.4 qC
47.5 CH
36.8 qC
23.2 CH2
122.9 CH
142.9 qC
41.5 qC
0.96 (m); 1.51 (m)
1.79 (m); 1.82 (m)
3.42 (dd, 11.0, 4.9)
27
28
29
25.5 CH3
175.2 qC
106.8 CH2
95.2 CH
73.3 CH
78.1 CH
70.4 CH
77.3 CH
69.0 CH2
1.17 (s)
4.63 (s); 4.68 (s)
6.21 (d, 8.2)
4.09 (m)
4.18 (t, 9.0)
4.31 (t, 9.0 )
4.05 (m)
4.36 (dd, 11.4, 4.8)
4.73 (br d, 11.4)
5.02 (d, 7.6)
Glc-11
21
0.82 (d, 11.9)
1.36 (m); 1.51 (m)
1.33 (m); 1.45 (m)
31
41
51
1.60 (dd, 10.9, 6.9)
61
1.86 (m); 1.89 (m)
5.40 (t, 3.6)
Glc-111
211
104.7 CH
74.9 CH
78.1 CH
70.8 CH
74.5 CH
64.0 CH2
4.02 (m)
4.18 (t, 9.0)
4.11 (m)
4.01 (m)
311
411
27.7 CH2
23.0 CH2
46.8 qC
2.31 (m)
2.02 (m); 2.12 (m)
511
611
4.88 (dd, 11.8, 5.5)
5.03 (dd, 11.8, 1.6)
47.0 CH
41.1 CH2
147.8 qC
29.5 CH2
37.1 CH2
28.2 CH3
16.0 CH3
15.1 CH3
17.0 CH3
3.10 (dd, 13.4, 5.1)
2.15 (m); 2.55 (t, 13.4)
Caff-1111
2111
126.4 qC
115.3 CH
147.0 qC
149.8 qC
116.1 CH
121.6 CH
145.4 CH
114.4 CH
167.1 qC
7.54 (br s)
3111
2.08 (m); 2.17 (m)
1.75 (m); 2.03 (m)
1.20 (s)
4111
5111
7.17 (d, 8.2 )
7.09 (dd, 8.2, 1.9)
7.91 (d, 15.8)
6111
1.01 (s)
0.91 (s)
1.09 (s)
7111
8111
6.61 (d, 15.8)
9111
δ in ppm; 1H-NMR: 600 MHz and 13C-NMR 150 MHz.
Table 3. NMR spectroscopic data for compound 3 in pyridine-d5.
No.
δc
δ
(mult., J in Hz)
No.
δc
δ (mult., J in Hz)
H
H
4.25 (d, 10.0); 4.36 (d,
10.0)
Ag-1
38.9 CH2
0.92 (m); 1.45 (m)
24
73.1 CH2
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
28.3 CH2
79.3 CH
43.0 qC
56.5 CH
19.2 CH2
33.3 CH2
39.5 qC
47.9 CH
37.0 qC
23.7 CH2
122.3 CH
144.2 qC
41.8 qC
28.1 CH2
23.7 CH2
46.6 qC
44.2 CH
47.9 CH2
69.7 qC
36.0 CH2
34.9 CH2
23.3 CH3
1.86 (m); 1.93 (m)
3.50 (dd, 4.1, 11.7)
25
26
27
28
29
15.6 CH3
17.0 CH3
25.8 CH3
180.1 qC
25.5 CH3
0.84 (s)
0.91 (s)
1.17 (s)
0.88 (br d, 11.7)
1.49 (m); 1.63 (m)
1.19 (m); 1.35 (m)
1.55 (s)
Glc-11
105.6 CH
74.9 CH
78.2 CH
71.2 CH
75.3 CH
64.3 CH2
4.93 (d, 7.7)
4.02 (dd, 8.3, 7.7)
4.21 (dd, 8.4, 8.3)
4.11 (dd, 9.6, 8.4)
4.10 (m)
1.56 (m)
21
31
41
51
61
1.82 (m)
5.49 (br s)
4.95 (dd, 11.4, 4.7)
5.03 (br d, 10.1)
1.11 (m); 2.12 (m)
1.99 (m); 2.20 (m)
Caff-111
211
126.6 qC
115.7 CH
147.5 qC
150.0 qC
116.6 CH
121.9 CH
145.8 CH
114.6 CH
167.4 qC
7.49 (d, 1.6)
311
3.32 (dd, 14.3, 3.9)
1.88 (m); 2.40 (t, 13.5)
411
511
7.18 (d, 8.2)
7.05 (dd, 8.2, 1.6)
7.88 (d, 15.8)
611
1.08 (m); 2.02 (m)
2.03 (m)
711
811
6.54 (d, 15.8)
1.51 (s, 3H)
911
δ in ppm; 1H-NMR: 600 MHz and 13C-NMR 150 MHz.

Molecules 2016, 21, 734
5 of 8
It is noteworthy that the caffeoylation of nortriterpenoid saponins (
2
and
3) were isolated from
family Lardizabalaceae for the first time. This kind of esterification is particularly important for
saponins for increasing their solubility in water (and hence to favor its lymphatic transport), thus
the esterification of many secondary metabolites generally represents one of the last steps of their
biosynthesis [12]. Because noroleanane triterpenoids are recognized as characteristic constituents of
the family Lardizabalaceae [3], the occurrence of those caffeoylation derivatives in Holboellia coriacea
may have a role as chemotaxonomic markers for Holboellia, which is worthy of further study.
2.2. Cytotoxicity Assay
The cytotoxicity of compounds 1–5 against three human tumors HepG2, HCT116 and SGC-7901
cell lines were assessed, using a sulforhodamine B (SRB) method. The resulting IC50 values are
displayed in Table 4. Only compound
and SGC-7901 cell lines, with IC50 values of 9.1, 15.2 and 41.0
studies on this type of nortriterpenoids suggested that the exocyclic double bond at C-20(29) might
be an important active center to “maintain” their potential cytotoxicity [ ], while the present result
5
showed interesting cytotoxicity against HCT-116, HepG2
µM, respectively. Previous cytotoxicity
5
suggested that the saccharide unit attached at C-28 could greatly reduce the in vitro cytotoxicity.
Table 4. Cytotoxicity against Cancer Cell Lines of compounds 1–5 (IC50, µM).
Compounds
HepG2
HCT-116
SGC-7901
1
2
3
4
>100
>100
>100
>100
15.2
>100
>100
>100
>100
9.1
87.0
>100
>100
>100
41.0
5.1
5
Adriamycin
0.46
3.3
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were determined using a Perkin-Elmer 341 polarimeter (PerkinElmer Co.,
Waltham, MA, USA). Ultraviolet (UV) spectra were taken on an UVmini-1240 spectrometer
(Shimadzu Co., Kyoto, Japan) and HRESIMS spectra on an API QSTAR mass spectrometer (Applied
Biosystem/MSD Sciex, Concord, ON, Canada). The 1H, 13C, and 2D NMR spectra were recorded on
a Bruker Avance-600 or a Bruker DRX-400 instrument using TMS as an internal standard. Column
chromatography was performed on silica gel 60 (200–300 mesh, Qingdao Marine Chemical Ltd.,
Qingdao, China). For Preparative TLC plates (HSGF254, Jiangyou silicone Development Co., Ltd.,
Yantai, China), Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) and Develosil ODS (50
Chemical Co. Ltd., Osaka, Japan) were used.
µm, Nomura
3.2. Plant Material
The stems of H. coriacea were collected from Xiangxi, Hunan Province, China, in December
2012, and identified by Prof. Zhang Dai-gui (Key Laboratory of Plant Resources Conservation and
Utilization, Jishou University, Jishou, China). A voucher specimen (zdg-20121203) has been deposited
at the Hunan Agricultural University.
3.3. Extraction and Isolation
Dried stems of H. coriacea (5 kg) were powdered and extracted with 95% EtOH (3
each) at room temperature, then evaporated in vacuo to give 550 mg of residue. The residue was
further suspended in H2O (1 L) and sequentially extracted with petroleum ether (PE, 3 1 L) and
EtOAc (3 1 L), to yield a PE-soluble fraction (15.4 g) and an EtOAc-soluble fraction (61.8 g). The
ˆ
15 L, 48 h
ˆ
ˆ

Molecules 2016, 21, 734
6 of 8
EtOAc-soluble fraction was subjected to silica gel column chromatography (CC) (100–200 mesh) with
elution of CHCl3-MeOH (100:0 60:40, v/v) to give nine fractions (Fr. A1–A9). Fraction A2 was
Ñ
decolorized on MCI gel column eluting with a gradient solvent system consisting of 20%, 85% and
100% (MeOH-H2O, v/v). The fraction eluted with 85% MeOH was chromatographed on MPLC
(ODS-C18) using MeOH-H2O (50:50
Compound (28 mg) was obtained from sub-fraction A2-e which was purified by normal silica gel
CC (petroleum ether-EtOAc, 9:1), compound (16 mg) from sub-fraction A2-f was purified on a
Sephadex LH-20 column (MeOH), and compound (30 mg) was recrystallized from sub-fraction A2-e.
Similarly, Fractions A6 (6.0 g), A7 (6.0 g) and A8 (8.3 g) were fractionated by an ODS-C18 column with
elution of MeOH-H2O (50:50 100:0, v/v) system, respectively. Compound (30 mg) precipitated from
sub-fraction A6-f and compound (30 mg) was purified from sub-fraction A7-d by gel filtration on a
Ñ100:0, v/v) system, to produce seven sub-fractions (A2-a–A2-g).
6
7
5
Ñ
8
4
Sephadex LH-20 column (MeOH). Whereas, sub-fraction A8-d and A8-f were purified successively on
preparative TLC (GF254) plates using chloroform-methanol-water (10:3:1, v/v/v) as mobile solvent
and Sephadex LH-20 column using MeOH as eluent, to obtain 3 (10 mg), 1 (22 mg), and 2 (19 mg).
3.4. Spectroscopic Data of 1–3
25
Compound
1
: White solid powder, rαsD + 125.0 (c = 0.5, MeOH); IR (KBr)
υ
3415, 2930, 1741, 1679,
max
1433, 1385, 1133, 1047 cm´1; 1H-NMR (400 MHz, pyridine-d5) and 13C-NMR (100 MHz, pyridine-d5)
spectroscopic data, see´Table 1; positive ion ESIMS m/z: 787 [M + Na]+; negative ESIMS m/z: 763
[M ´ H] , 799 [M + Cl] ; HR-ESIMS m/z: 787.4248 [M + Na]+ (calcd for C41H64O13 Na, 787.4239).
´
25
Compound
(4.00), 242 (2.32), 129 (2.94); IR (KBr)
2
: light yellow powder, rαsD + 25.7 (c = 2.3, MeOH); UV (MeOH)
λ
nm (log ε) 345
max
υ
max 3435, 3420, 3414, 2933, 2854, 1745, 1689, 1620, 1451, 1435, 1380,
1045 cm´1; 1H-NMR (600 MHz, pyridine-d5) and 13C-NMR (125 MHz, pyridine-d5) spectroscopic data,
see Table 1; positive ion ESIMS m/z: 949 [M + Na]+; negative ESIMS m/z: 961 [M + Cl]´; HR-ESIMS
´
m/z: 961.4378 [M + Cl] (calcd for C50H70O16Cl, 961.4358).
25
Compound
(2.59), 244 (1.78), 217 (2.41); IR (KBr)
3
: light yellow powder, rαsD + 45.0 (c = 2.0, MeOH); UV (MeOH)
λ
nm (log
ε
) 329
max
1
υ
3435, 3069, 1719, 1648, 1621, 1298, 1073 cm´1; H-NMR
max
(600 MHz, pyridine-d5) and 13C-NMR (125 MHz, pyridine-d5) spectroscopic data, see Table 1; positive
ion ESIMS m/z: 8´21 [M + Na]+; negative ESIMS m/z: 797 [M
´
H]´, 833 [M + Cl]´; HR-ESIMS m/z:
797.4109 [M ´ H] (calcd for C44H61O13, 797.4118).
3.5. Chemical Hydrolysis of 1–3
Alkaline and Acid Hydrolysis of
(3 mL) was stirred at 85 ˝C for 4 h. The reaction mixture was acidified to pH 4.0 with 10% HCl and
extracted with CHCl3 (3 10 mL). The CHCl3 layer was dried (anhydr. MgSO4) and concentrated
under reduced pressure. The residue was subjected to Sephadex LH-20 CC using MeOH to afford
akebonoic acid (1a) for the aglycone (5 mg from , 3.5 mg from
). 1a was identified on the basis of 1H
and 13C-NMR spectrum comparisons with that of published data for akebonoic acid [
]. The aqueous
layer was further concentrated and the residue in 5%–10% H2SO4 (2 drops) was heated in a boiling
H2O bath for 1 h. The solution was extracted with n-BuOH (3 20 mL). The n-BuOH solution was
1 and 2: A solution of saponins (1 or 2, 10 mg each) in 5% KOH
ˆ
1
2
7
ˆ
concentrated in vacuo and desalted (Sephadex LH-20, MeOH) to afford the sugar residue (1.0 mg).
˝
The sugar residue was derivatized with Sigma Sil-A for 35 min at 70 C and analyzed by GC-MS with
only one peak detected (Rt = 12.19 min, EI-MS m/z: M+ 540) [14], which was identified as trimethylsilyl
derivative of D-glucose.
Alkaline and Acid Hydrolysis of 3: Compounds 3 (8.0 mg) in 1 N HCl (5 mL, 1,4-dioxane-H2O,
1:1) was heated under reflux for 8 h. After removal of the solvent, the residue was partitioned
between CHCl3 and H2O. The CHCl3-soluble portion was evaporated and subjected to ODS column
chromatography using 90% MeOH to yield 4 mg of the aglycone (3a) which was identified as

Molecules 2016, 21, 734
7 of 8
3
β
,20
α
,24-trihydroxy-29-norolean-12-en-28-oic acid [13]. By the method described in
1
and
2, the
sugar unit of 3 was identified as D-glucose.
1
Compound 1a: H-NMR (600 MHz, pyridine-d5)
δ
5.51 (1H, br t, H-12), 4.80/4.76 (2H, each s, H2-29,
3.60 (1H, dd, J = 9.6, 5.8 Hz, H-3), 3.25 (1H, dd, J = 13.0, 4.9 Hz, H-18), 1.25 (6H, s, H3-23/ H3-24), 1.03
(3H, s, H3-27), 1.01 (3H, s, H3-26), 0.88 (3H, s, H3-25); 13C-NMR (150 MHz, Pyr)
δ
39.3 (C-1), 28.4 (C-2),
78.4 (C-3), 39.7 (C-4), 56.1 (C-5), 19.1 (C-6), 33.6 (C-7), 40.1 (C-8), 48.4(C-9), 37.7 (C-10), 24.2 (C-11),
123.3 (C-12), 144.5 (C-13), 42.4 (C-14), 28.6 (C-15), 24.1 (C-16), 47.4 (C-17), 48.3 (C-18), 42.4 (C-19), 149.5
(C-20), 38.7 (C-21), 30.8 (C-22), 29.1 (C-23), 16.9 (C-24), 15.9 (C-25), 17.7 (C-26), 26.5 (C-27), 179.9 (C-28),
107.4 (C-29).
1
Compound 3a: H-NMR (600 MHz, pyridine-d5)
δ 5.57 (1H, brs, H-12), 4.50 (1H, d, J = 10.8 Hz, H-23),
3.65/3.64(2H, m, H-3/H-23), 3.38 (1H, dd, J = 13.6, 5.5Hz, H-18), 1.60 (3H, s, H3-29), 1.56 (3H, s,
H3-24),1.26 (3H, s, H3-27), 0.99 (3H, s, H3-26), 0.86 (3H, s, H3-25); 13C-NMR (150 MHz, Pyr)
δ
38.7 (C-1),
28.3 (C-2), 80.2 (C-3), 43.2 (C-4), 56.4 (C-5), 19.1 (C-6), 33.6 (C-7), 39.8 (C-8), 48.1 (C-9), 37.1 (C-10), 23.9
(C-11), 122.6 (C-12), 144.4 (C-13), 42.1 (C-14), 28.4 (C-15), 28.4 (C-16), 46.8 (C-17), 44.4 (C-18), 48.2 (C-19),
69.9 (C-20), 36.3 (C-21), 35.2 (C-22), 23.6 (C-23), 64.5 (C-24), 16.0 (C-25), 17.0 (C-26), 26.0 (C-27), 180.7
(C-28), 25.7 (C-29).
3.6. Bioassay
The cytotoxicity of compounds 1–5 against three human cancer cell lines, human gastric carcinoma
(SGC-7901), human colon carcinoma (HCT 116) and human liver hepatocellular carcinoma (HepG2)
were assayed at the National Center for Drug Screening, Shanghai, China. Sulforhodamine B (SRB)
(Sigma-Aldrich Chemie GmbH, Munich, Germany) was used to test the effects of the compounds on
cell growth and viability [15]. Adriamycin was used as the positive control. All tests were performed
in triplicate, and results are expressed as IC50 values.
4. Conclusions
The present work 1r1 eported three new nortriterpenoid saponins, akebonoic acid
1
28-O-
B (
that the two caffeoylation of nortriterpenoidal saponins (
Lardizabalaceae for the first time. 3 -akebonoic acid ( ) showed interesting cytotoxicity against
HCT-116, HepG2 and SGC-7901 cell lines, with IC50 values of 9.1, 15.2 and 41.0 M, respectively.
β
-D-glucopyranosyl-(1
Ñ
6 )-
β
-D-glucopyranosyl ester (
1
), Holboelliside A (
2
) and Holboelliside
3
) isolated from the stem of Holboellia coriacea, along with five known compounds. It is noteworthy
and ) were isolated from family
2
3
α
5
µ
In addition, the saccharide unit attached at C-28 of this type of nortriterpenoids may greatly reduce
the in vitro cytotoxicity.
Supplementary Materials: The HR-ESI-MS, 1H- and 13C-NMR and 2D-NMR spectra (Figures S1–S21) of
compounds 1–3 are available online at www.mdpi.com/1420-3049/21/6/734/s1.
Acknowledgments: We thank Zhang Dai-Gui (Key Laboratory of Plant Resources Conservation and Utilization,
Jishou University) for the collection and authentication of plant material. This work was supported by the National
Nature Science Foundation of China (No. 31400308) and the Research Foundation of Education Bureau of Hunan
Province, China (No. 14C0569).
Author Contributions: In this paper, Guanhua Li, Ye Li and Hualiang He performed the extraction and isolation
of the compounds; Zhiwen Li performed the bioassays; Wenbing Ding carried out the structural elucidations and
the manuscript writing; and Youzhi Li designed and coordinated the study and reviewed the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
Flora of China Editorial Committee. Flora of China; Science Press: Beijing, China, 2001; Volume 29, p. 13.
Zhu, Z.; Chen, T.; Pi, H.F.; Ruan, H.L.; Wu, J.Z.; Zhang, P. A new triterpenoid saponin and other saponins
from the roots of Holboellia coriacea. Chem. Nat. Compd. 2015, 51, 89–893. [CrossRef]

Molecules 2016, 21, 734
8 of 8
3.
Zhen, Q.A.; Yang, C.R. Chemotaxonomic study on the family of Lardizabalaceae. Chin. Bull. Bot. 2001, 18,
332–339.
4.
5.
Ying, Q.; Liang, J.Y.; Feng, X. Research in nor-oleanane triterpenoids. Nat. Prod. Res. Dev. 2011, 23, 577–581.
Wang, J.; Xu, Q.L.; Zheng, M.F.; Ren, H.; Lei, T.; Wu, P.; Zhou, Z.Y.; Wei, X.Y.; Tan, J.W. Bioactive
30-noroleanane triterpenes from the pericarps of Akebia trifoliata. Molecules 2014, 19, 4301–4312. [CrossRef]
[PubMed]
6.
Murakami, T.; Oominami, H.; Yoshikawa, M. Bioactive saponins and glycosides. XVIII. Nortriterpene
and triterpene oligoglycosides from the fresh leaves of Euptelea polyandra Sieb. et ZUCC.(2): Structures of
eupteleasaponins VI, VI Acetate, VII, VIII, IX, X, XI, and XII. Chem. Pharm. Bull. 2001, 49, 741–746. [CrossRef]
[PubMed]
7.
8.
9.
Ikuta, A.; Itokawa, H. Triterpenoids of Akebia quinata callus tissue. Phytochemistry 1986, 25, 1625–1628.
[CrossRef]
Ikuta, A.; Itokawa, H. A triterpene from Akebia quinata callus tissue. Phytochemistry 1988, 27, 3809–3810.
[CrossRef]
Ikuta, A.; Itokawa, H. Triterpenoids of Paeonia japonica callus tissue. Phytochemistry 1988, 27, 2813–2815.
[CrossRef]
10. Ikuta, A.; Itokawa, H. 30-Noroleanane saponins from callus tissues of Akebia quinata. Phytochemistry 1989, 28,
2663–2665. [CrossRef]
11. Gülcemal, D.; Masullo, M.; Senol, S.G.; Alankus¸-Çalis¸kan, Ö.; Piacente, S. Saponins from Cephalaria aristata C.
Koch. Rec. Nat. Prod. 2014, 8, 228–233.
12. Flamini, G.; Braca, A.; Cioni, P.L.; Morelli, I.; Tomè, F. Three new flavonoids and other constituents from
Lonicera implexa. J. Nat. Prod. 1997, 60, 449–452. [CrossRef]
13. Fu, H.; Koike, K.; Zheng, Q.; Mitsunaga, K.; Jia, Z.; Nikaido, T.; Lin, W.; Guo, D.; Zhang, L. Fargosides A-E,
triterpenoid saponins from Holboellia fargesii. Chem. Pharm. Bull. 2001, 49, 999–1002. [CrossRef] [PubMed]
14. Ding, W.B.; Huang, R.; Zhou, Z.S.; Li, Y.Z. New sesquiterpenoids from Ambrosia artemisiifolia L. Molecules
2015, 20, 4450–4459. [CrossRef] [PubMed]
15. Sun, Z.H.; Tan, N.H.; Zeng, G.Z.; Zhang, Y.M. Two new cinnamyl isovalerate derivatives from Sabina gaussenii.
Molecules 2016, 21, 571–576. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 6–8 are available from the authors.
©
2016 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
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).