Erythrosaponins A–J, triterpene saponins from the roots and stem bark of Gardenia erythroclada

Article abstract of DOI:10.1016/j.phytochem.2018.04.016

Ten undescribed triterpene saponins, named erythrosaponins A–J, along with one known analogue were isolated from the roots and stem bark of Gardenia erythroclada. Their structures were determined on the basis of extensive 1D and 2D NMR analyses. Absolute structure of erythrosaponin A was unequivocally affirmed by single-crystal X-ray crystallography. All isolated compounds were evaluated for their cytotoxicity against cancer cell lines (KB and HeLa S-3) and their anti-inflammatory activity based on the inhibition of NO production in RAW264.7 cells. Erythrosaponin D showed moderate cytotoxicity against KB and HeLa S-3 cells with IC₅₀ values of 25.8 and 29.5 μM, respectively. Erythrosaponins D, F, G, I and J showed moderate anti-inflammatory with IC₅₀ values in the range of 63.0–81.4 μM.

Full text of DOI:10.1016/j.phytochem.2018.04.016

Phytochemistry 152 (2018) 36e44
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Erythrosaponins AeJ, triterpene saponins from the roots and stem
bark of Gardenia erythroclada
a
a
b
b
Sutin Kaennakam , Thammarat Aree , Jantana Yahuafai , Pongpun Siripong ,
Santi Tip-pyang
a
a, *
Center of Excellence in Natural Products Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand
Natural Products Research Section, Research Division, National Cancer Institute, Bangkok, 10400, Thailand
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 December 2017
Received in revised form
Ten undescribed triterpene saponins, named erythrosaponins AeJ, along with one known analogue were
isolated from the roots and stem bark of Gardenia erythroclada. Their structures were determined on the
basis of extensive 1D and 2D NMR analyses. Absolute structure of erythrosaponin A was unequivocally
affirmed by single-crystal X-ray crystallography. All isolated compounds were evaluated for their cyto-
toxicity against cancer cell lines (KB and HeLa S-3) and their anti-inflammatory activity based on the
inhibition of NO production in RAW264.7 cells. Erythrosaponin D showed moderate cytotoxicity against
23 April 2018
Accepted 26 April 2018
KB and HeLa S-3 cells with IC50 values of 25.8 and 29.5
showed moderate anti-inflammatory with IC50 values in the range of 63.0e81.4
2018 Elsevier Ltd. All rights reserved.
m
M, respectively. Erythrosaponins D, F, G, I and J
Keywords:
Gardenia erythroclada
Rubiaceae
m
M.
©
Triterpene saponins
Erythrosaponins AeJ
1
. Introduction
parts (roots and stem bark) of G. erythroclada. Cytotoxicity against
KB (human epidermoid carcinoma) and Hela Se3 (human cervix
adenocarcinoma) cells and anti-inflammatory activity based on the
inhibition of NO production in RAW264.7 cells were evaluated for
all isolated compounds.
Gardenia erythroclada Kurz (Rubiaceae) (synonym Dioecrescis
erythroclada), also known as “Ma Khang Daeng” in Thai, is a tree
widely distributed in the northeastern part of Thailand. It is used
for traditional Thai medicine to relieve abdominal pain and stom-
achache, and for antipyretic purposes. Iridoid glycosides, aromatic
glycosides and aliphatic glycosides have been reported from the
leaves and branches of this plant (Kaewkrud et al., 2007). The genus
Gardenia comprises more than 130 species distributed over tropical
and subtropical regions of Asia, Africa, Australia, Madagascar and
some Pacific Islands (Mai et al., 2016). This genus has interesting
pharmacological activities such as anticancer, antibacterial, anti-
implantation, antioxidant, anti-inflammatory and anti HIV prop-
erties (Youn et al., 2016). Previous phytochemical investigation on
this genus revealed the presence of triterpenoids (Nuanyai et al.,
2. Results and discussion
The methanol extracts of dried roots (10.0 kg) and stem bark
(10.0 kg) of G. erythroclada were subjected to multiple chromato-
graphic steps over Dianion HP-20, silica gel and Sephadex LH-20,
yielding compounds 1e5, 8, 9 and 11 from the roots part and
compounds 6e10 from the stem bark part. A total of eleven tri-
terpene saponins were obtained, including ten undescribed com-
pounds, erythrosaponins AeJ (1e10) and one known compound,
catunaroside G (11) (Gao et al., 2011). The structures of 1e11 (Fig. 1)
were identified by physical data analyses, including 1D and 2D
NMR, IR, HRESIMS and single-crystal X-ray crystallography. The
sugar residues were identified by co-TLC, HPLC analyses and optical
rotation after hydrolysis.
2
009), flavonoids (Bhandare and Laddha, 2016), iridoids (Xiao
et al., 2017) and phenolic compounds (Wang et al., 2016). In this
report, we discuss the isolation and structural elucidation of ten
undescribed triterpene saponins, erythrosaponins AeJ (1e10) and
one known analogue (11) from the methanol crude extracts of two
Erythrosaponin A (1) was obtained as colorless crystals with
2
0
½
aꢀ -39.7 (c 0.26, MeOH). Its molecular formula was determined as
D
þ
C
C
42
H
H
68
O
13 from the ion peak [MþNa] at m/z 803.4567 (calcd. for
42
68
O
13Na, 803.4558) in the positive HRESIMS. The IR spectrum
*
Corresponding author.
ꢁ1
E-mail address: santi.ti@chula.ac.th (S. Tip-pyang).
showed absorption bands at 3428, 1727, and 1645 cm , suggesting
https://doi.org/10.1016/j.phytochem.2018.04.016
031-9422/© 2018 Elsevier Ltd. All rights reserved.
0

S. Kaennakam et al. / Phytochemistry 152 (2018) 36e44
37
Fig. 1. Chemical structures of 1e11.
the presence of hydroxyl, carbonyl, and olefinic groups, respec-
carbon at
carbon at
d
C
90.6 (C-3), the anomeric proton at
d
H
5.35 and the
1
13
tively. The H and C NMR data of 1 (Table 1) exhibited signals for
seven methyl groups at 0.74 (3H, s, H-26), 0.85 (3H, s, H-24), 0.92
6H, s, H-25, 29), 0.94 (3H, s, H-30), 1.04 (3H, s, H-23), and 1.28 (3H,
d
C
79.2 (C-2-GlcI), and between the oxymethine proton at
105.8 (C-1-Glc) and
102.0 (C-1-Rha) confirmed the glucose residue was linked to C-3
d
H
d
H
3.41 (1H, m, H-2-GlcI) and the carbons at d
C
(
d
C
s, H-27), one olefinic proton at
typical olefinic carbon signals at
d
H
5.29 (1H, br s, H-12) with two
125.2 (C-12) and 144.8 (C-13),
of aglycone and the terminal rhamnose was substituted at 2-OH of
1
13
d
C
the glucopyranosyl unit. The comparison of the H and C NMR
spectroscopic data (Table 1) of 1 showed similar resonances to
those of 11, apart from the lack of a glucopyranosyl unit at C-28 of
aglycone. Absolute structure of 1 based on its b-D-glucose moiety
was also affirmed by single-crystal X-ray crystallography (Fig. 3).
On the basis of the above results, the structure of compound 1 was
which were characteristic of an olean-12-ene skeleton. The spec-
troscopic data also showed signals of two oxymethines at 3.16
1H, dd, J ¼ 3.9,11.6 Hz, H-3) with 90.6 (C-3) and at 3.23 (1H, d,
J ¼ 3.2 Hz, H-19) with 82.7 (C-19), and a carbon of carboxylic acid
at 182.5 (C-28). The NOESY correlations (Fig. 2) between H-3 and
H-23, and between H-19 and H-30 indicated the -orientation of H-
-orientation of H-19. Thus, the aglycone was identified as
-dihydroxyolean-12-ene-28-oic acid (siaresinolic acid)
d
H
(
d
C
d
H
d
C
d
C
a
identified as 3-O-[
anosyl]-3 ,19 -dihydroxyolean-12-en-28-oic acid.
Erythrosaponin B (2) was obtained as an amorphous powder
a-L-rhamnopyranosyl-(1/2)-b-D-glucopyr-
3
3
and
,19
b
b
a
b
a
2
0
(
Barton et al., 1952). The presence of two sugar residues was
confirmed from the observation of two anomeric protons at 4.39
1H, d, J ¼ 7.3 Hz, H-1-GlcI) and 5.35 (1H, br s, H-1-Rha) (Table 3),
which were correlated in the HSQC spectrum with anomeric car-
bons at 105.8 and 102.0, respectively. After acid hydrolysis, the
sugar units were confirmed to be -glucose and -rhamnose, which
with ½aꢀ -33.3 (c 0.20, MeOH). Its molecular formula of C42
68 14
H O
D
þ
d
H
was established by the positive HRESIMS [MþNa] ion peak at m/z
1
(
819.4500 (calcd. for C42
H
68
O14Na, 819.4507). Comparing the H and
13
C NMR data (Tables 1 and 3) of 2 with those of 1 showed the same
d
C
sugar components and sequence of sugar chains. The difference
D
L
was in the aglycone part; the methyl group at C-23 of aglycone in 1
1
13
were identified by co-TLC, HPLC analyses and optical rotation. The
configuration of the glucose and rhamnose glycosidic bonds was
was replaced to a hydroxymethyl group. The H and C NMR
spectra showed two anomeric protons at
4.51 (1H, d, J ¼ 7.7 Hz,
H-1-GlcI) and 5.34 (1H, br s, H-1-Rha), which were correlated in the
HSQC spectrum with anomeric carbons at 104.6 and 101.7,
respectively. The NMR spectra of 2 showed two proton signals at
d
H
established as
coupling constants (Table 3) and the NOESY correlations. The HMBC
correlations (Fig. 2) between the anomeric proton at 4.39 and the
b-glucose and a-rhamnose, respectively by the
d
C
d
H
d
H

38
S. Kaennakam et al. / Phytochemistry 152 (2018) 36e44
Table 1
1
H and 13C NMR Spectroscopic Data for the Aglycones of 1e5 (MeOD-d
4
).
Position
1
2
3
4
5
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
1.00, m; 1.56, m
1.69, m; 1.93, m
3.16, dd (3.9, 11.6)
40.1
27.4
90.6
40.4
57.6
19.7
34.2
40.9
49.2
38.2
25.0
125.2
144.8
42.8
29.7
28.7
46.9
45.4
82.7
36.2
29.7
34.2
28.9
0.98, m; 1.55, m
1.73, m; 1.97, m
3.64, dd (3.6, 10.7)
39.5
26.6
82.2
43.9
48.6
18.9
33.3
40.6
49.2
37.7
24.7
124.9
144.5
42.6
29.4
28.6
46.6
45.1
82.4
35.9
29.4
34.0
64.6
1.03, m; 1.61, m
1.75, m; 1.99, m
3.68, dd (3.5, 12.1)
39.7
26.7
82.3
43.9
48.7
19.0
33.3
40.8
49.3
37.7
24.8
125.0
144.3
42.7
29.4
28.5
47.1
45.1
82.5
35.9
29.5
33.4
64.7
1.03, m; 1.58, m
1.70, m; 1.99, m
3.21, d (3.4, 11.6)
40.1
27.3
90.7
40.5
57.6
19.7
33.8
41.0
49.5
38.2
25.0
125.2
144.7
42.8
29.8
28.7
47.0
45.4
82.7
36.2
29.7
34.3
28.7
1.03, m; 1.58, m
1.69, m; 1.93, m
3.21, dd (3.4, 11.8)
39.9
27.2
90.1
40.2
57.4
19.4
34.0
40.7
49.4
38.0
24.8
125.0
144.5
42.5
29.5
28.5
46.7
45.1
82.5
36.0
29.4
34.0
28.5
0.79, m
1.42, m; 1.55, m
1.27, m; 1.50, m
1.26, m
1.39, m; 1.49, m
1.22 m; 1.60, m
1.32, m
1.42, m; 1.53, m
1.28, m; 1.64, m
0.81, m
1.22, m; 1.58, m
1.31, m; 1.54, m
0.79, m
1.42, m; 1.56, m
1.29, m; 1.51, m
1.72, m
1.77, m
1.81, m
1.75, m
1.71, m
0
1
2
3
4
5
6
7
8
9
0
1
2
3
1.92, m
5.29, br s
1.95, m
5.30, br s
1.98, m
5.36, br s
1.95, m
5.33, br s
1.93, m
5.31, br s
1.00, m; 1.72, m
2.27, m
1.00, m; 1.74, m
2.27, m
1.04, m; 1.74, m
2.35, m
1.02, m; 1.78, m
2.31, m
0.99, m; 1.76, m
2.25, m
3.03, br s
3.23, d (3.2)
3.04, br s
3.24, d (3.6)
3.08, br s
3.31, br s
3.07, br s
3.27, d (3.4)
3.04, br s
3.26, br s
1.60, m
1.60, m; 1.76, m
1.04, s
1.59, m
1.60, m; 1.75, m
3.36, d (11.3);
1.68, m
1.61, m
1.64, m; 1.79, m
1.07, s
1.62, m
1.61, m; 1.77, m
1.04, s
1.67, m; 1.81, m
3.35, d (11.5);
3.63, d (11.5)
0.75, s
1.00, s
0.78, s
3.59, d (11.3)
2
2
2
2
2
2
3
4
5
6
7
8
9
0
0.85, s
0.92. s
0.74, s
1.28, s
17.3
16.1
18.0
25.3
182.5
28.9
25.4
0.71, s
0.95, s
0.76, s
1.30, s
13.7
16.2
17.7
25.0
182.3
28.6
25.1
13.7
16.3
17.8
25.0
178.6
28.6
25.2
0.88, s
0.96, s
0.78, s
1.31, s
17.4
16.1
18.0
25.2
182.5
28.9
25.4
0.86, s
0.93, s
0.75, s
1.28, s
17.2
15.9
17.7
25.1
182.4
28.6
25.2
1.34, s
0.92, s
0.94, s
0.93, s
0.95, s
0.98, s
0.99, s
0.95, s
0.98, s
0.93, s
0.95, s
Fig. 2. Key HMBC (arrow curves) and NOESY (dashed curves) correlations of 1 and 6.
3
.36 (1H, d, J ¼ 11.3 Hz, H-23a) and 3.59 (1H, d, J ¼ 11.3, H-23b) with
carbon signal at 64.6 (C-23) instead of a methyl signal in 1. In
addition, the HMBC correlations of H-23 with C-3 ( 82.2), C-5 (
8.6), and C-24 ( 13.7) also confirmed the presence of the
hydroxymethyl group at C-23. Thus, the aglycone of 2 was identi-
fied as ,19 ,23 -trihydroxyolean-12-en-28-oic acid (ilex-
osapogenin A) (Amimoto et al., 1993). Finally, the structure of 2 was
78
for C48H O19Na, 981.5035). According to the NMR analysis and
d
C
hydrolysis results, compound 3 possessed the same aglycon and 3-
O-sugar chain as 2, except that the carboxylic acid group was
d
C
d
C
1
13
4
d
C
substituted with a glucopyranosyl group. The H and C NMR
spectra showed three anomeric protons at
d
H
4.55 (1H, d, J ¼ 7.5 Hz,
3b
a
a
H-1-GlcI), 5.39 (1H, br s, H-1-Rha), and 5.41 (1H, d, J ¼ 8.1 Hz, H-1-
Glc), which were correlated in the HSQC spectrum with anomeric
assigned
anosyl]-3
as
,19
3-O-[
a
-
L
-rhamnopyranosyl-(1/2)-
b
-
D
-glucopyr-
carbons at
trum showed cross-peak between the anomeric proton at
(1H, d, J ¼ 8.1 Hz, H-1-Glc) and the carbon at 178.6 (C-28), sug-
gesting that the -glucose was located at C-28 of 2. Accordingly, 3
was determined to be 3-O-[ -rhamnopyranosyl-(1/2)-b-D-
d
C
104.6, 101.8, and 95.8, respectively. The HMBC spec-
b
a
,23
a-trihydroxyolean-12-en-28-oic acid.
d
H
5.41
Erythrosaponin C (3) was obtained as an amorphous powder
d
C
2
0
with ½aꢀ -74.7 (c 0.54, MeOH). Its molecular formula was deter-
b-D
D
mined to be C48
H
78
O
19 by HRESIMS [MþNa]þ m/z 981.5044 (calcd.
a-
L

S. Kaennakam et al. / Phytochemistry 152 (2018) 36e44
39
2
Fig. 3. ORTEP drawing of 1, Note that the glucose moiety has a doubly disordered C6' (H )eOH group with occupancy factors of 0.56 and 0.44.
ꢁ
1
glucopyranosyl]-28-O-(
yolean-12-en-28-oic acid.
Erythrosaponin D (4) was obtained as an amorphous powder
b
-
D
-glucopyranosyl)-3
b
,19
a
,23
a
-trihydrox-
spectrum showed absorption bands at 3426, 1725, and 1642 cm
,
suggesting the presence of hydroxyl, carbonyl, and olefinic groups,
1
13
respectively. The H and C NMR data of 6 (Table 1) showed signals
of seven methyl groups at 0.84 (3H, s, H-24), 0.84 (3H, s, H-25),
0.84 (3H, s, H-26), 0.93 (3H, br s, H-29), 0.94 (3H, br s, H-30), 1.02
20
with ½aꢀ -103.1 (c 0.32, MeOH). Its molecular formula of C42
H
66
O
14
d
H
D
þ
was suggested by the positive HRESIMS [MþNa] ion peak at m/z
1
13
8
17.4349 (calcd. for C42
H
66
O
14 Na, 817.4350). The H and C NMR
(3H, s, H-23), and 1.09 (3H, s, H-27), one olefinic proton at 5.23
d
H
data of 4 were similar to those of 1, except for a sugar unit. The
(1H, br s, H-12) with two typical olefinic carbon signals at d 127.2
C
hydroxymethyl group of glucopyranosyl moiety was oxidized to
(C-12) and 139.1 (C-13), which were characteristic of an urs-12-ene
skeleton. The NMR spectra also exhibited a signal for an oxy-
become a carboxylic acid group (
moiety. The HMBC correlations observed from
H-5-GlcA) to 105.8 (C-1-GlcA), 79.3 (C-3-GlcA), and 176.0 (C-6-
GlcA) confirmed the presence of a glucuronic acid unit. Two
anomeric proton signals at
4.47 (1H, d, J ¼ 7.0 Hz, H-1-GlcA) and
.39 (1H, br s, H-1-Rha) were correlated in the HSQC spectrum with
anomeric carbons at 105.8 and 102.1, respectively. On the basis of
the above deductions, the structure of 4 was established as 3-O-[
-rhamnopyranosyl-(1/2)- -glucuronopyranosyl]-3 ,19 -dihy-
droxyolean-12-en-28-oic acid.
Erythrosaponin E (5) was obtained as an amorphous powder
d
C
176.0) in glucuronopyranosyl
d
H
3.64 (1H, d, J ¼ 8.1,
methine at
and one carbonyl carbon at
(Fig. 2) showed cross-peak at H-3 with H-23, at H-18 (
J ¼ 11.2 Hz) with H-20 ( 0.94, m) and H-29 indicated the
entations of H-3 and H-19, and -orientation of H-20. Thus, the
aglycone part was identified as 3 -hydroxyurs-12-en-28-oic acid
(ursolic acid) (Varanda et al., 1992). Four anomeric proton signals at
d
H
3.19 (1H, dd, J ¼ 3.2, 11.4 Hz, H-3) with
178.0 (C-28). The NOESY correlations
2.21, d,
-ori-
C
d 90.6 (C-3),
d
C
d
C
d
H
d
H
d
H
a
5
b
d
C
b
a-
L
b-
D
b
a
d
H
4.44 (1H, d, J ¼ 7.5 Hz, H-1-GlcI), 4.52 (1H, d, J ¼ 7.8 Hz, H-1-
GlcII), 5.33 (1H, d, J ¼ 8.1 Hz, H-1-Glc), and 5.44 (1H, br s, H-1-
Rha) were observed, which were correlated in the HSQC spec-
2
0
with ½aꢀ -82.5 (c 0.20, MeOH). Its molecular formula was deter-
trum with anomeric carbons at
respectively. After acid hydrolysis, the sugar units were confirmed
to be -glucose and -rhamnose. The configuration of the glucose
and rhamnose was established by the coupling constants as
glucose and
The HMBC correlations (Fig. 2) between the anomeric proton at
4.44 (H-1-GlcI) with the carbon at
proton at 4.52 (H-1-GlcII) with the carbon at
the anomeric proton 5.44 (H-1-Rha) with the carbon at
(C-2-GlcI), the anomeric proton at 5.33 (H-1-Glc) with the car-
bon at 178.0 (C-28), and between the oxymethine proton at
3.58 (1H, m, H-2-GlcI) with the carbon at 88.5 (C-3-GlcI), 101.7
(C-1-Rha), and 105.3(C-1-GlcI) confirmed the -rhamnopyr-
anosyl-(1/2)- -glucopyranosyl-(1/3)- -glucopyranosyl
chain at 3-OH and -glucopyranosyl moiety at 28-COOH of
aglycone. On the basis of the above results, the structure of 6 was
identified as 3-O-[ -rhamnopyranosyl-(1/2)- -glucopyr-
anosyl-(1/3)- -glucopyranosyl]-28-O-( -glucopyranosyl)-
-hydroxyurs-12-en-28-oic acid.
Erythrosaponin G (7) was obtained as an amorphous powder
C
d 105.3, 103.9, 95.6, and 101.7,
D
þ
mined as C48
H
78
O
18 from the positive HRESIMS [MþNa] ion peak
at m/z 965.5083 (calcd. for C48
78
H O
18Na, 965.5086). Comparison of
the H and C NMR spectra of 5 with those of 1 indicated that 5
differed from 1 in the presence of an additional deoxyglucose unit.
D
L
1
13
b
-
a
-rhamnose (Table 4) and the NOESY experimental.
Three anomeric proton signals at
d
H
4.45 (1H, d, J ¼ 7.6 Hz, H-1-
d
H
GlcI), 4.53 (1H, d, J ¼ 7.8 Hz, H-1-GlcII), and 5.44 (1H, br s, H-1-
d
C
90.6 (C-3), the anomeric
88.5 (C-3-GlcI), at
77.9
Rha) were correlated in the HSQC spectrum with anomeric car-
d
H
d
C
bons at
showed cross-peak between the anomeric proton at
carbon at 88.5 (C-3-GlcI), between the oxymethine proton at
.60 (H-2-GlcI) and the carbons at 88.5, 101.7 (C-1-Rha), and
05.3 (C-1-GlcI) confirmed the presence of a -rhamnopyranosyl-
1/2)- -glucopyranosyl-(1/3)- -glucuronopyranosyl chain
located at C-3 of 5. Thus, the structure of 5 was determined to be 3-
O-[ -rhamnopyranosyl-(1/2)- -glucopyranosyl-(1/3)-
glucopyranosyl]-3 ,19 -dihydroxyolean-12-en-28-oic acid.
Erythrosaponin F (6) was obtained as an amorphous powder
d
C
105.3, 103.9, and 101.7, respectively. The HMBC spectrum
d
H
d
C
d
H
4.53 and the
d
H
d
C
d
H
d
C
d
H
3
d
C
d
C
1
a-
L
a-L
(
b-
D
b
-D
b-
D
b-D
b-D
a-L
b
-D
b-D-
b
a
a-L
b-D
b-
D
b-D
2
0
with ½aꢀ -97.5 (c 0.54, MeOH). Its molecular formula was deter-
3b
D
þ
mined to be C54
H
88
O
O
22 from the ion peak [MþNa] at m/z 1111.5660
2
0
(
calcd. for C54
H
88
22Na, 1111.5665) in the positive HRESIMS. The IR
with ½aꢀD -101.8 (c 0.38, MeOH). Its molecular formula of C54
88 22
H O

40
S. Kaennakam et al. / Phytochemistry 152 (2018) 36e44
Table 2
1
H and 13C NMR Spectroscopic Data for the Aglycones of 6e9 (MeOD-d
4 6
) and 10 (DMSO‑d ).
Position
6
7
8
9
10
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1.02, m; 1.62, m
1.68, m; 1.91, m
3.19, dd (3.2, 11.4)
40.2
27.2
90.1
42.5
57.3
19.3
34.3
40.9
49.3
37.9
24.4
127.2
139.1
43.2
29.3
25.2
49.5
54.2
40.3
40.2
31.7
37.8
28.5
17.6
17.3
17.9
24.0
178.0
16.3
21.6
1.06, m; 1.60, m
1.71, m; 1.95, m
3.21, dd (3.3, 11.9)
37.8
27.1
90.1
41.0
57.3
19.3
34.3
40.2
49.3
37.8
24.4
127.3
139.0
43.2
29.3
25.2
49.5
54.2
40.4
40.2
31.7
37.5
28.6
17.7
17.3
17.9
24.0
178.0
16.3
21.6
1.02, m; 1.60, m
1.68, m; 1.90, m
3.18, dd (3.7, 11.4)
38.5
27.4
91.2
41.2
57.4
19.7
34.7
40.3
49.4
38.2
24.8
127.2
140.0
43.6
29.6
25.7
48.9
54.7
40.7
40.5
32.1
38.2
29.0
18.1
17.5
18.2
24.6
182.0
16.5
22.0
1.09, m; 1.70, m
1.73, m; 1.96, m
3.24, dd (3.0, 10.7)
37.8
27.0
90.9
40.0
57.0
19.3
34.3
40.1
49.0
40.0
24.4
127.2
139.0
43.2
29.2
25.2
49.2
54.1
40.3
40.2
31.7
37.4
28.6
17.7
17.1
17.9
24.1
177.9
16.2
21.6
0.86, m; 1.55, m
1.50, m; 1.93, m
3.07, m
39.2
25.8
88.2
41.9
55.6
18.0
32.9
39.7
47.6
36.1
23.1
125.2
137.9
41.9
27.9
23.4
47,4
52.6
38.6
38.4
30.3
36.5
27.7
17.0
16.6
17.3
23.4
175.4
15.7
21.3
0.75, m
1.37, m; 1.51, m
1.31, m; 1.48, m
0.76, m
1.36, m; 1.53, m
1.31, m; 1.53, m
0.77, m
1.37, m; 1.53, m
1.32, m; 1.52, m
0.81, m
1.43, m; 1.57, m
1.37, m; 1.55, m
0.71, m
1.45, m
1.23, m; 1.44, m
1.52, m
1.52, m
1.53, m
1.57, m
1.44, m
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.90, m
5.23, br s
1.91, m
5.23, br s
1.91, m
5.21, br s
1.97, m
5.29, br s
1.83, m
5.14, br s
1.08, m; 1.91, m
1.78, m; 2.05, m
1.10, m; 1.93, m
1.76, m; 2.04, m
1.10, m; 1.90, m
1.78, m; 2.02, m
1.16, m; 1.98, m
1.79, m; 2.12, m
0.89, m; 1.85, m
1.64, m; 1.96, m
2.21, d (11.2)
1.37, m
0.94, m
1.31, m; 1.48, m
1.61, m; 1.73, m
1.02, s
0.84, s
0.84, s
0.84, s
1.09, s
2.22, d (11)
1.39, m
0.97, m
1.31, m; 1.52, m
1.62, m; 1.75, m
1.04, m
0.82, m
0.82, m
0.82, m
1.10, m
2.18, d (11.2)
1.35, m
0.96, m
1.32, m; 1.48, m
1.66, m; 1.78, m
1.05, s
0.83, s
0.83, s
0.83, s
1.10, s
2.27, d (11.1)
1.43, m
1.01, m
1.38, m; 1.55, m
1.68, m; 1.79, m
1.12, s
0.88, s
0.88, s
0.88, s
1.16, s
2.10, dd (10.0)
1.33, m
0.92, m
1.24, m; 1.46, m
1.50, m; 1.64, m
0.95, s
0.82, s
0.76, s
0.72, s
1.02, s
0.93, br s
0.94, br s
0.94, br s
0.96, br s
0.94, br s
0.94, br s
1.00, br s
1.00, br s
0.87, br s
0.92, br s
Table 3
1
H and 13C NMR Spectroscopic Data for the Sugar Units of 1e5 (MeOD-d
4
).
Position
1
2
3
4
5
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
3
1
2
3
4
5
6
-O-
b
-D-GlcI
b
-D-GlcI
b
-D-GlcI
b
-D-GlcA
b
-D-GlcI
4.39, d (7.3)
3.41, m
3.21, m
3.27, m
3.43, m
105.8
79.2
77.8
72.3
79.7
63.0
4.51, d (7.7)
3.36, m
3.23, m
3.27, m
3.46, m
104.6
78.7
77.4
72.0
79.3
62.7
4.55, d (7.5)
3.42, m
3.29, m
3.34, m
3.51, m
104.6
78.8
78.3
72.0
79.3
62.8
4.47, d (70)
3.51, m
3.51, m
3.51, m
3.64, d (8.1)
105.8
79.1
79.3
74.2
76.9
176.0
4.45, d (7.6)
3.60, m
3.71, m
3.28, m
3.48, m
105.3
78.0
88.5
71.5
78.2
62.7
3.64, dd (5.1, 11.8)
3
3.64, dd (5.2, 11.7)
3.83, dd (1.3, 11.7)
3.71, dd (5.2, 11.6)
3.87, dd (1.8, 11.6)
3.66, dd (5.0, 11.5)
3.88, dd (1.5, 11.5)
.83, dd (1.3, 11.8)
b-D-GlcII
1
2
3
4
5
6
4.53, d (7.8)
3.55, m
3.29, m
3.38, m
3.37, m
103.9
73.9
77.3
71.9
78.1
62.5
3.66, dd (5.0, 11.5)
3.88, dd (1.5, 11.5)
a-L-Rha
a-L-Rha
a-L-Rha
a-L-Rha
a-L-Rha
1
2
3
4
5
6
2
1
2
3
4
5
6
5.35, br s
3.93, m
3.73, dd (3.1, 9.5)
3.37, m
3.97, m
102.0
72.2
72.4
74.2
70.2
18.2
5.34, br s
3.94, m
3.74, dd (3.3, 9.5)
3.37, m
3.95, m
101.7
71.8
72.1
73.9
69.9
17.9
5.39, br s
4.01, m
3.79, dd 2.9, 9.5)
3.37, m
3.99, m
1.26, d (6.0)
b-D-Glc
5.41, d (8.1)
3.41, m
3.63, m
3.38, m
101.8
71.9
72.1
73.9
70.0
17.9
5.39, br s
3.98, m
3.77, dd (3.2, 9.5)
3.40, t (9.6)
3.99, m
102.1
72.3
72.4
74.2
70.2
18.2
5.44, br s
3.98, m
3.74, m
3.39, m
4.01, m
101.7
72.0
72.1
73.7
70.1
18.0
1.20, d (6.1)
1.21, d (6.2)
1.23, d (6.1)
1.21, d (6.1)
8-O-
95.8
74
78.7
71.1
77.4
62.4
3.29, m
3.71, dd (5.2, 11.6)
3.87, dd (1.8, 11.6)

S. Kaennakam et al. / Phytochemistry 152 (2018) 36e44
41
Table 4
1
H and 13C NMR Spectroscopic Data for the Sugar Units of 6e9 (MeOD-d
4 6
) and 10 (DMSO‑d ).
Position
6
7
8
9
10
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
3
1
2
3
4
5
6
-O-
b
-D-GlcI
b
-D-Gal
b
-D-GlcI
b
-D-GlcI
b-D-GlcA
4.32, d (5.5)
3.44, m
3.64, m
3.11, m
4.44, d (7.5)
3.58, m
3.71, m
3.29, m
3.37, m
105.3
77.9
88.5
71.5
78.2
62.7
4.48, d (6.8)
3.66, m
3.78, m
3.35, m
3.36, m
3.67, m
3.79, m
105.3
78.0
86.6
71.2
78.2
62.4
4.37, d (7.8)
3.39, m
3.53, m
3.39, m
3.48, m
3.67, m
3.86, m
106.6
74.3
88.4
70.4
77.6
63.1
4.43, d (7.7)
3.43, m
3.57, m
3.41, m
4.40, m
3.72, m
3.89, m
106.2
73.9
88.0
70.0
77.9
62.7
103.7
75.5
85.7
69.8
77.4
172.8
3.13, m
3.67, m
3
.83, m
b
-D-GlcII
b-D-GlcII
b
-D-GlcII
b-D-GlcII
b-D-GlcII
1
2
3
4
5
6
4.52, d (7.8)
3.30, m
3.29, m
3.34, m
3.37, m
103.9
73.8
77.2
71.1
78.1
62.6
4.58, d (7.4)
3.32, m
3.32, m
3.35, m
3.36, m
3.67, m
3.86, m
103.4
73.9
78.0
71.2
78.2
62.5
4.58,d (7.8)
3.52, m
3.54, m
3.39, m
3.35, m
3.67, m
3.86, m
105.4
75.4
84.4
70.4
78.4
63.0
4.63, d (7.8)
3.43, m
3.58, m
3.41, m
3.40, m
3.72, m
3.89, m
105.0
75.9
84.0
70.0
78.2
62.6
4.43, d (5.5)
3.10, m
3.44, m
3.11, m
3.20, m
3.47, m
3.71, m
102.0
72.5
75.2
69.8
76.9
61.1
3.67, m
3.83, m
a-L-Rha
a-L-Rha
a-L-Rha
a-L-Rha
a-L-Rha
1
2
3
4
5
6
2
1
2
3
4
5
6
5.44, br s
3.97, m
3.73, m
3.38, m
4.01, m
101.7
72.0
72.1
73.7
70.1
18.0
5.42, br s
3.99, m
3.74, m
3.37, m
3.99, m
101.7
71.9
72.1
73.7
70.2
18.0
5.17, br s
3.95, m
3.71, m
3.53, m
3.98, m
103.0
72.7
72.6
74.3
70.5
18.3
5.22, br s
3.99, m
3.75, m
3.56, m
4.03, m
102.6
72.2
72.1
73.8
70.1
17.9
5.33, br s
3.77, m
3.47, m
3.19, m
3.86, m
100.4
70.5
70.6
72.2
68.5
18.0
1.20, d (6.1)
1.21, d (6.0)
1.24, d (6.2)
1.30, d (6.1)
1.05, d (4.7)
b-D-Glc
8-O-
b
-D-Glc
b-D-Glc
b-D-Glc
5.33, d (8.1)
3.26, m
3.69, m
3.35, m
3.29, m
95.6
75.2
78.5
70.1
77.2
62.5
5.34, d (8.1)
3.28, m
3.68, m
3.35, m
3.33, m
3.67, m
3.86, m
95.7
75.1
78.5
71.3
78.0
62.5
5.40, d (8.0)
3.38, m
3.64, m
3.41, m
3.34, m
3.72, m
3.89, m
95.6
74.9
78.4
71.1
77.2
62.5
5.18, d (8.1)
3.06, m
3.13, m
3.11, m
3.20, m
3.49, m
3.60, m
94.3
73.8
77.8
70.2
76.9
61.1
3.67, m
3.83, m
þ
was established by the positive HRESIMS [MþNa] ion peak at m/z
111.5651 (calcd. for C54 22 Na, 1111.5665). Careful comparison
5.17 (1H, br s, H-1-Rha), which were correlated in the HSQC spec-
trum with anomeric carbons at 106.6, 105.4, and 103.0, respec-
tively. The cross-peak between the anomeric protons at 4.37 (H-
1-Glc1) and the carbon at 91.2 (C-3), the anomeric proton at
4.58 (H-1-GlcII) and the carbon at 88.4 (C-3-GlcI), and between
the anomeric proton at 5.17 (H-1-Rha) and the carbon at 84.4
(C-3-GlcII) in the HMBC spectrum suggested that the -rhamno-
pyranosyl-(1/3)-O-[O- -glucopyranosyl-(1/3)]- -glucopyr-
anosyl was located at C-3 ( 91.2) and the trisaccharide chain was
also confirmed by comparison of the NMR data with the literature
(Lemmich et al.,1995). Consequently, 8 was deduced to be 3-O-{
rhamnopyranosyl-(1/3)-O-[O- -glucopyranosyl-(1/3)]-
glucopyranosyl}-3 -hydroxyurs-12-en-28-oic acid.
Erythrosaponin I (9) was obtained as an amorphous powder
1
H
88
O
d
H
of the NMR spectroscopic data of 7 with those of 6 indicated no
difference between two compounds, except for the replacement of
the glucopyranosyl by galactopyranosyl unit. Acid hydrolysis of 7
d
H
d
C
d
H
d
C
yielded a sugar mixture containing
D
-glucose,
rhamnose. The 1H and C NMR spectra showed four anomeric
protons at
D
-galactose, and
L-
d
H
d
C
13
a-L
d
H
4.48 (1H, d, J ¼ 6.8 Hz, H-1-Gal), 4.58 (1H, d, J ¼ 7.4 Hz,
b
-
D
b-D
H-1-GlcII), 5.34 (1H, d, J ¼ 8.1 Hz, H-1-Glc), and 5.42 (1H, br s, H-1-
d
C
Rha, which were correlated in the HSQC spectrum with anomeric
carbons at
d
C
105.3, 103.4, 95.7, and 101.7, respectively. The HMBC
spectrum showed cross-peak between the anomeric proton at
a-L-
d
H
b
-D
b-D-
4
.48 and the carbon at
d
C
90.1 (C-3), the anomeric proton at
d
H
4.58
5.42
b
and the carbon at
and the carbon at
proton at
01.7 (C-1-Rha), and 105.3 (C-1-Gal) confirmed the presence of
rhamnopyranosyl-(1/2)- -glucopyranosyl-(1/3)- -gal-
d
C
86.6 (C-3-Gal), the anomeric proton at d
H
2
0
d
C
78.0 (C-2-Gal), and between the oxymethine
86.6 (C-3-Gal),
with ½aꢀ -74.7 (c 0.50, MeOH). Its molecular formula of C54
88 22
H O
D
þ
d
H
3.66 (H-2-Gal) and the carbon at
d
C
was suggested by the positive HRESIMS [MþNa] ion peak at m/z
1
13
1
a-L-
1111.5670 (calcd. for C54
88
H O22 Na, 1111.5665). The H and C NMR
b
-D
b-D
data of 9 were similar to those of 8, except for the presence of a 28-
1
13
actopyranosyl unit and the sugar chain was also compared with the
NMR data of literature (Gupta et al., 2000). Thus, the structure of 7
O-glucopyranosyl unit. The H and C NMR spectra showed four
anomeric protons at
d
H
4.43 (1H, d, J ¼ 7.7 Hz, H-1-GlcI), 4.63 (1H, d,
was elucidated as 3-O-[
pyranosyl-(1/3)- -galactopyranosyl]-28-O-(
anosyl)-3 -hydroxyurs-12-en-28-oic acid.
Erythrosaponin H (8) was obtained as an amorphous powder
a-
L
-rhamnopyranosyl-(1/2)-
b
-D
-gluco-
J ¼ 7.8 Hz, H-1-GlcII), 5.22 (1H, br s, H-1-Rha), and 5.40 (1H, d,
b
-D
b-D-glucopyr-
J ¼ 8.0 Hz, H-1-Glc), which were correlated in the HSQC spectrum
b
C
with anomeric carbons at d 106.2, 105.0, 102.6, and 95.6, respec-
tively. In addition, acid hydrolysis of 9 also confirmed the presence
of -glucose and -rhamnose. Thus, the structure of 9 was eluci-
dated as 3-O-{ -rhamnopyranosyl-(1/3)-O-[O- -glucopyr-
anosyl-(1/3)]- -glucopyranosyl}-28-O-( -glucopyranosyl)-
-hydroxyurs-12-en-28-oic acid.
Erythrosaponin J (10) was obtained as an amorphous powder
2
0
with ½aꢀ -115.2 (c 0.66, MeOH). Its molecular formula was deter-
D
L
D
þ
mined to be C48
H
78
O
17 by HRESIMS [MþNa] m/z 949.5142 (calcd.
a-L
b-D
for C48H
78
O
17Na, 949.5137). The comparison of the spectroscopic
b
-D
b-D
data of 8 with those of 6 (Tables 2 and 4) showed similarity, except
for the absence of sugar unit at C-28 and the position of ramno-
pyranosyl in 6 was located from C-2-GlcI to C-3-GlcII in 8. The H
and C NMR spectra showed three anomeric protons at
3b
1
20
with ½aꢀD -24.1 (c 0.58, DMSO). Its molecular formula was deter-
13
þ
d
H
4.37
mined to be C54
H
86
O
23 from the positive HRESIMS [MþNa] ion
(
1H, d, J ¼ 7.8 Hz, H-1-GlcI), 4.58 (1H, d, J ¼ 7.8 Hz, H-1-GlcII), and
86
peak at m/z 1125.5468 (calcd. for C54H O23Na, 1125.5458). Analysis

42
S. Kaennakam et al. / Phytochemistry 152 (2018) 36e44
of H and 13C NMR data (Tables 2 and 4) of 10 showed similarity to
those of 6 except that the glucopyranosyl unit (GlcI) of 6 was
replaced by a glucuronopyranosyl unit, the signal of carboxyl group
1
3.3. Extraction and isolation
The dried powder of G. erythroclada roots (10.0 kg) and stem
bark (10.0 kg) were macerated for 3 days with MeOH (each
2 ꢂ 25 L). After evaporation of the solvent in vacuo, 450 g of root
residue and 420 g of stem bark residue were obtained. Each of the
two MeOH residue samples were suspended in H
separated with dianion HP-20, and eluted with H
respectively.
The MeOH-soluble residue (98 g) from the roots was chroma-
tographed over silica gel and eluted with MeOH in EtOAc (0e100%,
stepwise), yielding four fractions (A-D). Fraction A (5 g) was puri-
C
shown at d 172.8 (C-6-GlcA) and the lack of a hydroxymethyl group
in 10. The H and C NMR spectra showed four anomeric protons at
1
13
d
H
4.32 (1H, d, J ¼ 5.5 Hz, H-1-GlcA), 4.43 (1H, d, J ¼ 5.5 Hz, H-1-
GlcII), 5.33 (1H, br s, H-1-Rha), and 5.18 (1H, d, J ¼ 8.1 Hz, H-1-
2
O and then
O and MeOH,
Glc), which were correlated in the HSQC spectrum with anomeric
2
carbons at
drolysis confirmed the presence of a sugar mixture containing
glucose, -glucoronic acid, and -rhamnose. The NMR spectroscopic
data (Table 4) of 10 confirmed the presence of -glucoronic acid
moiety. In addition, the sugar sequence was elucidated by the 2D
NMR data. Thus, 10 was identified as 3-O-[ -rhamnopyranosyl-
1/2)- -glucopyranosyl-(1/3)- -glucuronopyranosyl]-28-
O-( -glucopyranosyl)-3 -hydroxyurs-12-en-28-oic acid.
All isolated compounds (1e11) were evaluated in vitro for their
C
d 103.7, 102.0, 100.4, and 94.3, respectively. Acid hy-
D
-
D
L
b
fied with sephadex LH-20, using CH
give 1 (120 mg). Fraction B (12 g) was repeatedly subjected to silica
gel, eluting with MeOH in CH Cl (1:4), to provide two subfractions,
and then each subfraction was further purified with sephadex LH-
20, using CH Cl -MeOH (1:1) as eluent to afford 2 (95 mg) and 8
(25 mg). Compounds 5 (35 mg) and 11 (45 mg) were achieved from
fraction C (10 g) by siliga gel, eluting with CH Cl -MeOH (4:1).
Fraction D (15 g) was separated by silica gel and eluted with CH Cl
2 2
Cl -MeOH (1:1) as eluent to
a-L
(
b-
D
b-
D
2
2
b
-D
b
2
2
cytotoxicity against KB and HeLa S-3 cell lines and their anti-
inflammatory activity based on the inhibition of NO production in
RAW264.7 cells. Almost all compounds showed inactive cytotox-
icity against two cancer cells, except that compound 4 showed
moderate cytotoxicity against KB and HeLa S-3 cells with IC50
2
2
2
2
-
MeOH (4:1) to obtain two subfractions (Da and Db). Subfraction Da
(5 g) was further purified with sephadex LH-20, eluting with
values of 25.8 and 29.5
oleanane-type saponins have the 28-carboxylic acid based on the
damage of cell membrane like a hemolytic action (Podolak et al.,
m
M, respectively. The cytotoxic effects of
CH
(15 mg) was purified from subfraction Db (2 g) with sephadex LH-
20, eluting with CH Cl -MeOH (1:1).
2 2
Cl -MeOH (1:1), to yield 3 (15 mg) and 4 (11 mg). Compound 9
2
2
2
010). In this research suggested that disaccharide at C-3 of ole-
The MeOH-soluble residue (85 g) from the stem bark was sub-
jected to silica gel and eluted with MeOH in EtOAc (0e100%, step-
wise), to provide three fractions (E-G). Compound 8 (10 mg) was
purified from fraction E (3 g) with sephadex LH-20, eluting with
anane triterpenes, glucuronic acid-rhamnose (4) have more cyto-
toxic effect than glucose-rhamnose (1) and trisaccharide (5).
Compounds 4, 6, 7, 9 and 10 showed moderate anti-inflammatory
with IC50 values of 76.8, 81.4, 69.4, 63.0 and 79.5
All isolated compounds have no affected to RAW264.7 cells at
concentration lower than 300 M. Doxorubicin (IC50 0.16 and
.03 M for KB and HeLa S-3) was used for positive control of
cytotoxicity and dexamethasone (IC50 8.25 mM) was used for posi-
tive control of anti-inflammatory.
m
M respectively.
CH
silica gel and eluted with a gradient of CH
give 6 (10 mg) and 9 (8 mg). Fraction G (17 g) was fractionated by
siliga gel and eluted with CH Cl -MeOH (2:1), to give two sub-
fraction (Ga and Gb). Compound 7 (12 mg) was purified from
subfraction Ga (5 g) with sephadex LH-20, eluting with CH Cl
MeOH (1:1). Subfraction Gb was treated with sephadex LH-20,
using CH Cl -MeOH (1:1) as eluent to yield 10 (9 mg).
2
Cl
2
-MeOH (1:1). Fraction F (20 g) was chromatographed over
2
2
Cl -MeOH (1:4 to 4:1), to
m
0
m
2
2
2
2
-
2
2
3
. Experimental section
3
.3.1. Erythrosaponin A (1)
2
0
3.1. General experimental procedures
Colorless crystals; ½aꢀ -39.7 (c 0.26, MeOH); IR (KBr)
nmax 3428,
D
ꢁ1
1
13
1727, 1645 cm ; H and C NMR data, see Tables 1 and 3; positive
þ
The UVevisible absorption spectra were recorded on a UV-2550
HRESIMS m/z 803.4567 [Mþ Na] (calcd. for
42 68
C H O13Na,
UVevis spectrometer (Shimadzu, Kyoto, Japan). The IR spectra were
measured on a Nicolet 6700 FT-IR spectrometer using KBr discs.
Optical rotations were detected by a Jasco P-1010 polarimeter. NMR
spectra were recorded on a Bruker 400 AVANCE spectrometer
803.4558).
3.3.2. Erythrosaponin B (2)
2
0
Amorphous powder; ½aꢀ -33.3 (c 0.20, MeOH); IR (KBr)
n
max
D
1
13
ꢁ1
1
13
(
400 MHz for H and 100 MHz for C). The HRESIMS were obtained
3425, 1729, 1647 cm
;
H and C NMR data, see Tables 1 and 3;
þ
using a Bruker MICROTOF model mass spectrometer. Single-crystal
positive HRESIMS m/z 819.4500 [Mþ Na] (calcd. for C42
68
H O14Na,
X-ray diffraction analysis was performed on a Bruker ꢂ8 APEXII
819.4507).
Kappa CCD area-detector diffractometer with MoK
a radiation
(
l
¼ 0.71073 Å). Column chromatography was performed with
3.3.3. Erythrosaponin C (3)
2
0
Dianion HP-20, silica gel 60 (0.063e0.200 mm), and Sephadex LH-
2
Amorphous powder; ½aꢀ -74.7 (c 0.54, MeOH); IR (KBr)
n
max
D
ꢁ
1
1
13
0 (25e100
mm, GE Healthcare).
3421, 1725, 1646 cm
;
H and C NMR data, see Tables 1 and 3;
þ
positive HRESIMS m/z 981.5044 [Mþ Na] (calcd. for C42
68
H O13Na,
981.5035).
3.2. Plant material
3
.3.4. Erythrosaponin D (4)
2
0
The roots of Gardenia erythroclada Kurz (synonym Dioecrescis
Amorphous powder; ½aꢀ -103.13 (c 0.32, MeOH); IR (KBr)
n
max
D
ꢁ1
1
13
erythroclada) (Rubiaceae) were collected in July 2015 and the stem
bark of G. erythroclada was collected in January 2016 from Sahat-
3423, 1727, 1642 cm
;
H and C NMR data, see Tables 1 and 3;
þ
positive HRESIMS m/z 817.4349 [Mþ Na] (calcd. for C42
66
H O14Na,
ꢃ
0
00
ꢃ
0
00
sakhan district, Kalasin province, Thailand (16 43 35 N 103 29 22
817.4350).
E). The plant material was identified by Dr. Suttira Khumkratok, a
botanist at the Walai Rukhavej Botanical Research Institute,
Mahasarakham University, and a specimen retained as a reference
3.3.5. Erythrosaponin E (5)
2
0
Amorphous powder; ½aꢀ -82.5 (c 0.20, MeOH); IR (KBr)
n
max
D
ꢁ1
1
13
(
Khumkratok no. 5e12).
3431, 1730, 1648 cm
; H and C NMR data, see Tables 1 and 3;

S. Kaennakam et al. / Phytochemistry 152 (2018) 36e44
43
þ
positive HRESIMS m/z 965.5083 [Mþ Na] (calcd. for C48
H
78
O
18Na,
The crystal data have been deposited with the Cambridge Crystal-
lographic Data Centre (CCDC 1547305 and 1554048). Copies of
these data can be obtained, free of charge, on application to the
CCDC via ww.ccdc.cam.ac.uk/conts/retrieving.html (or 12 Union
Road, Cambridge CB2 1EZ, UK, fax: þ44 1223 336033, e-mail:
deposit@ccdc.cam.ac.uk).
9
3
3
65.5086).
.3.6. Erythrosaponin F (6)
2
0
Amorphous powder; ½aꢀ -97.5 (c 0.54, MeOH); IR (KBr)
n
max
D
ꢁ1
1
13
426, 1725, 1642 cm
;
H and C NMR data, see Tables 2 and 4;
þ
positive HRESIMS m/z 1111.5660 [Mþ Na] (calcd. for C54
88
H O22Na,
1111.5665).
3.5. Acid hydrolysis
3
.3.7. Erythrosaponin G (7)
A solution of erythrosaponins AeJ (1e10) (5 mg) in 2 M HCl
(1 mL) was heated at reflux for 24 h. The reaction mixture was
neutralized with 2 M NaOH and extracted by partition with EtOAc
(5 ꢂ 1 mL). The sugar residues were identified by co-TLC (TLC Silica
gel 60 F ) by comparison with standard sugar. The solvent system
2
0
Amorphous powder; ½aꢀ -101.8 (c 0.38, MeOH); IR (KBr)
n
max
D
ꢁ1
1
13
3
432, 1728, 1642 cm
;
H and C NMR data, see Tables 2 and 4;
þ
positive HRESIMS m/z 1111.5651 [Mþ Na] (calcd. for C54
88
H O22Na,
1111.5665).
2
54
was CH
2 2 2
Cl -MeOH-H O (2:1:0.2), and spots were visualized by
3
.3.8. Erythrosaponin H (8)
spraying with EtOH-H SO -anisaldehyde (9:0.5:0.5, v/v), then
heated at 150 C. The R values of
lonic acid and L-rhamnose by TLC were 0.30, 0.20, 0.04 and 0.56,
respectively. In addition, the sugar were identified by HPLC analysis
2
4
2
0
ꢃ
Amorphous powder; ½aꢀ -115.2 (c 0.66, MeOH); IR (KBr)
n
max
D-glucose,
D-galactose, D-gluco-
D
f
ꢁ1
1
13
3
426, 1724, 1640 cm
;
H and C NMR data, see Tables 2 and 4;
þ
positive HRESIMS m/z 949.5142 [Mþ Na] (calcd. for C48
H
17
O
22Na,
9
3
3
49.5137).
(column: RPM-Monosacc 300 ꢂ 7.8 mm, carrier: 100% H O 0.6 mL/
2
min, retention time: 13.37, 15.42, 15.77 and 25.05 min) in compar-
.3.9. Erythrosaponin I (9)
ison with the authentic D-glucose, L-rhamnose, D-galactose and D-
glucolonic acid, respectively. The optical rotation value was
2
D
0
Amorphous powder; ½aꢀ -74.7 (c 0.50, MeOH); IR (KBr)
n
max
ꢁ1
1
13
20
429, 1730, 1641 cm
;
H and C NMR data, see Tables 2 and 4;
measured after 24 h of dissolution in water, D-glucose ½aꢀ þ49.5 (c
D
þ
20
D
positive HRESIMS m/z 1111.5670 [Mþ Na] (calcd. for C54
H
88
O
22Na,
0.1, H O),
D
-galactose ½aꢀ þ69.2 (c 0.1, H O), D-glucolonic acid
2
2
2
0
20
D
1111.5665).
½aꢀ þ37.7 (c 0.1, H O) and L-rhamnose ½aꢀ -12.8 (c 0.1, H O)
D
2
2
(Tsukamoto et al., 1989), (Lehbili et al., 2017).
3
.3.10. Erythrosaponin J (10)
2
0
Amorphous powder; ½aꢀ -24.1 (c 0.58, DMSO); IR (KBr)
n
max
D
3.6. Cytotoxicity assay
ꢁ1
1
13
3
429, 1727, 1649 cm
;
H and C NMR data, see Tables 2 and 4;
þ
positive HRESIMS m/z 1125.5468 [Mþ Na] (calcd. for C54
86
H O23Na,
All isolated compounds (1e11) were subjected to cytotoxic
evaluation against KB (human epidermoid carcinoma) and HeLa S-3
1125.5458).
(
human cervical carcinoma) cell lines employing the colorimetric
3
.4. X-ray crystallographic analysis of 1
method (Kongkathip et al., 2003). Doxorubicin, which exhibits ac-
tivity against two cancer cell lines, was used as the reference
substance.
Colorless single crystals of 1 were obtained from the slow
evaporation of a MeOHeACN solution, as C42 13 in the mono-
clinic, space group P2 (no. 4) with unit cell constants
b ¼ 7.6106(5) Å, c ¼ 21.3562(15) Å,
calc ¼ 1.077 g/cm ,
68
H O
1
3
.7. Anti-inflammatory assay
a ¼ 15.0656(11)
Å,
ꢃ
3
3
b
¼ 100.306(2) , V ¼ 2409.2(3) Å , Z ¼ 2,
D
All isolated compounds (1e11) were subjected to anti-
MW ¼ 780.96. A rod-like single crystal of 1 with dimensions
.10 ꢂ 0.12 ꢂ 0.24 mm was fixed on the tip of MiTeGen microloop
using epoxy glue. With the help of a Bruker ꢂ8 APEXII Kappa CCD
area-detector diffractometer with MoK radiation (
¼ 0.71073 Å)
inflammatory activity based on the inhibition of NO production in
RAW264.7 cells using a previously published protocol (Chen et al.,
2
0
017). Dexamethasone was used as the positive control.
a
l
and the APEX2 Software Suite, X-ray diffraction data were collected
at 296(2) K. Data were then processed sequentially, i.e. integrated,
reduced by SAINTþ, corrected for Lorentz, polarization and ab-
sorption effects, and scaled by SADABS (SAINTþ and SADABS, 2008.
Bruker AXS Inc., Madison, Wisconsin, USA) to yield 8819 unique
reflections (Rint ¼ 0.067). The structure was solved using the
intrinsic phasing method with SHELXTL XT (Bruker, 2014b) and
Acknowledgements
The authors are grateful to the Graduate School of Chula-
longkorn University for
adaphiseksomphot Endowment Fund) to SK. We also thank Dr.
Suttira Khumkratok, Walai Rukhavej Botanical Research Institute,
Mahasarakham University, Mahasarakham 44000, Thailand for
identification and deposition of the plant material.
a Postdoctoral Fellowship (Ratch-
2
refined with full-matrix least squares on F with SHELXTL XTLMP
(Bruker, 2014a). The disordered solvents present in the large
intermolecular voids were removed using PLATON SQUEEZE pro-
Appendix A. Supplementary data
cedure (Spek, 2015). The refinement converged at the final
2
2
2
2
R(F ) ¼ 0.058 and wR(F ) ¼ 0.141 for 5115 data with F > 2
s(F ). The
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.phytochem.2018.04.016.
absolute structure of 1 was first established according to its
b-D-
glucose moiety with known absolute configuration (Fig.1) and then
directly re-confirmed by a Bruker ꢂ8 PROSPECTOR KAPPA CCD
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