New Triterpenoid Saponins from Green Vegetable Soya Beans and Their Anti-Inflammatory Activities

Article abstract of DOI:10.1021/acs.jafc.7b04134

Ten compounds were isolated and identified from green vegetable soya beans, of which five are new triterpenoid saponins (1-5) and five are known compounds (6-10). The chemical structures of the five triterpenoid saponins (1-5) were elucidated to be 3β,24-

Full text of DOI:10.1021/acs.jafc.7b04134

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Article
New Triterpenoid Saponins from Green Vegetable
Soya Beans and Their Anti-inflammatory Activities
Xiuhua Lan, Kejun Deng, Jianping Zhao, Yiyi Chen, Xuhui Xin, Yanli
Liu, Ikhlas A Khan, Shilin Yang, Taoyun Wang, and Qiong Ming Xu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04134 • Publication Date (Web): 29 Nov 2017
Downloaded from http://pubs.acs.org on November 30, 2017
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Page 1 of 24
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New Triterpenoid Saponins from Green Vegetable Soya Beans and Their
Anti-inflammatory Activities
†
, ‡
†
§
†
†
‡
Xiuhua Lan , Kejun Deng , Jianping Zhao , Yiyi Chen , Xuhui Xin , Yanli Liu ,
‡
, §
†
‡, #*
‡*
Ikhlas A. Khan , Shilin Yang , Taoyun Wang , Qiongming Xu
†
Department of Biotechnology, School of Life Science and Technology, Center for
Informational Biology, University of Electronic Science and Technology of China,
Chengdu, 610054, PR China
‡
College of Pharmaceutical Science, Soochow University, Suzhou, 215123, China
§
National Center for Natural Products Research, Department of Pharmacognosy,
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Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of
Mississippi, University, Mississippi, 38677
#
College of Chemical, Biological and Material Engineering, Suzhou Science and
Technology University, Suzhou, 215009, China
Corresponding Authors:
Qiongming Xu & Taoyun Wang
Phone: 86-512-69561421, 86-512-65882080
Fax: 86-512-65882089
E-mail: xuqiongming@suda.edu.cn; wangtaoyun@mail.usts.edu.cn
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ABSTRACT Ten compounds were isolated and identified from green vegetable soya
beans, of which five triterpenoid saponins (1-5) are new, and five compounds (6-10)
are known. The chemical structures of five triterpenoid saponins (1-5) were elucidated
to
3-O-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyl-(1→2)-β-D-glucuronopyranosid
e, 1, 3β,24-dihydroxy-22β,30-epoxy-30-oxoolean-12-en
3-O-α-L-rhamnopyranosyl-(1→2)-β-D-(3′′-O-formyl)-galactopyranosyl-(1→2)-β-D-gl
ucuronopyranoside, 2, 22-keto-3β,24-dihydroxy oleanane-12-ene
3-O-α-L-rhamnopyranosyl-(1→2)-β-D-(3′′-O-formyl)-galactopyranosyl-(1→2)-β-D-gl
ucuronopyranoside, 3, 3β,22β,24-trihydroxy oxyolean-18(19)-ene-29-acid
3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyrano
side, 4, and punicanolic acid
be
3β,24-dihydroxy-22β,30-epoxy-30-oxoolean-12-en
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3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyrano
side, 5, in terms of spectroscopic data (IR, GTC/FID, HR-ESI-MS, 1D and 2D NMR).
The nitric oxide release inhibitions of compounds 1-10 in LPS-stimulatedRAW264.7
cells were evaluated, and the data suggested that compounds 1, 2, 5 might possess
moderate anti-inflammatory activities, with the IC50 values of 18.8, 16.1, and 13.2 µM,
respectively.
KEYWORDS: soya beans; Glycine max; triterpenoid saponin; structure elucidation;
anti-inflammatory activities
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INTRADUCTION
Soybean plant [Glycine max (L.) Merr., Fabaceae], one of the most important healthy
food and economic crop, is cultivated in many countries and regions. So far,
following the United States, Brazil, and Argentina, China has ranked as the fourth
1
most productive country in annual gross production of soybean. Green vegetable
soya beans, known as maodou in China and edamame in Japan, are commonly
consumed as snack food and vegetable for dishes in many Asian countries.
According to the previous studies, green vegetable soya bean is a cornucopia of plant
protein, essential amino acid, ω-3 fatty acids, and various micronutrients that could
4
-6
exert series of physiological functions to humans and livestock.
Recent studies
7
8
9
10
indicated that isoflavones, saponins (soyasaponins), alkaloids, and steroids
contributed to the diverse bioactivities of soybeans. Soyasaponins, the major
1
1, 12
ingredients in the hypocotyl of soybean,
exhibit many biological activities, such
1
3
14
15
as cholesterol-lowering, renin-inhibiting, hepatoprotective, anti-kidney-disease
16
17, 18
19
20, 21
progression, antioxidative,
anti-inflammatory, and anti-tumor effects.
Some soyasponins, specifically soyasaponin Ab, have showed potential
2
2
anti-inflammatory activity and been the subject of intensive study. In this study, the
phytochemical investigation of saponins in green vegetable soya beans led to the
isolation of five new soyasaponins 1-5 (Figure 1), along with five known ones whose
structural elucidation and their anti-inflammatory activities are reported herein.
MATERIALS AND METHODS
1
13
General. Optical rotations, IR spectra, and H- and C-NMR were determined by a
polarimeter (PerkinElmer Inc., Waltham, MA), a spectrometer (PerkinElmer Inc.,
Waltham, MA), the 500 NMR facility (Varian Inc., Palo Alto, CA), respectively. The
Q-TOF MS/MS system (Micromass Corp., London, UK) was employed to detect the
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HR-ESI-MS data.
Chromatography. Two chromatographic columns were used. Column #1: 250×10.0
mm i.d., 5µm, Kromsil C18 (Akzo Nobel, Amsterdam, Holland). Column #2:
250×10.0 mm i.d., 5µm, Cosmosil cholester (Nacalai Tesque, INC., Kyoto, Japan).
The analysis was performed on column #1 or column #2, using the HPLC system
(Shimadzu Corp., Kyoto, Japan) with UV detection at 203 nm. Column #3: D101
macroporous resin column ( 200×30 cm i.d). and column #4: silica gel (60-100 mesh)
column (20×12 cm i.d) were used for pre-separation. A glass MPLC column #3 (460
mm × 26 mm i.d.) and an MPLC system including a C-650 pump (Büchi Corp.,
Flawil, Swiss) were used for purification ). The silica gel (60-100 mesh) and
precoated silica gel, supplied by Qingdao Marine Chemical Factory Co., Ltd, Qingdao,
China, were employed for raw material purification. 10% sulfuric acid ethanol
solution was employed as the color-developing agent for TLC. Sephadex LH-20 and
D101 macroporous resin were supplied by GE Corp. (Piscataway, NJ) and Prof.
Xiao-ran Li (Soochow, University), respectively. GC data were obtained by a GC-14C
system including a flame ionization detector (FID) (Shimadzu Corp., Kyoto, Japan).
The detailed analytical parameters were set as follows: DB-5 column, 30 m x 0.25
mm i.d column temperature at 210 ºC; injector temperature at 270 ºC; detector
temperature at 300 ºC. DB-5 column was a gift from Suzhou Huitong
Chromatography Technology Co., Ltd. Suzhou, China.
Materials.The fresh soya fruits were harvested in Suzhou area (Jiangsu, China)
during September 2012 and identified by Xiao-ran Li (Soochow University). In this
study, the flakelets of fresh fruits (5.0 kg) were dried under the sun. The voucher
specimen was stored in the herbarium of Soochow University and labeled as
No.12-09-16-01.
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The extraction and isolation process. The 1.2 kg sun-dried flakelets of soya fruits
was extracted two times by MeOH at 80 ºC under reflux. Subsequently, the MeOH
was recycled using the rotary evaporator to obtain a residue (225 g) that was
sequentially extracted by petroleum ether, CHCl , and EtOAc, respectively. Column
3
#3 was employed to purify the remaining water fraction, using a gradient of ethanol
(EtOH-H
was purified on column #4, using MeOH/CHCl
70%, 100%, each 3.0 L). The dried MeOH/CHCl
2
O 0-100, 30-70, 60-40, 90-10, each 30L). The dried 60-40 fraction (8 g)
(10%, 20%, 30%, 40%, 50%, 60%,
(20%) (1.2 g) was separated on
3
3
MPLC system with an ODS column , using MeOH-H O (60:40, 70:30, 80:20, 90:10,
2
100:0, each 300 mL) to obtain 5 fractions. Fraction 2 (525 mg) was purified on
column #1 using semi-preparative HPLC system and the flow rate of the mobile phase
(30% MeOH ) was at 2 min/ml, to yield compounds 6 (113 mg), 8 (48 mg) and 9 (53
mg), whose retention time were 25.04, 29.47, and 26.82 min, respectively. The
MeOH/CHCl (30%) fraction (0.9 g) was further purified by using MPLC with an
3
reverse-phase ODS column and eluting with MeOH/H O (50%, 60%, 70%, 80%
2
100%, each 300 mL) to give 5 fractions, of which Fraction 3 was further separated
using semi-preparative HPLC with column #1, eluting with 70% MeOH at the flow of
2 min/ml to obtain compounds 3 (12 mg) and 10 (29 mg), whose retention time were
33.05 and 30.53 min, respectively. Fraction 4 (108 mg) was further separated using
semi-preparative HPLC with column #1, eluting with 65% MeOH at 2 min/ml to give
R R
compound 7 (55 mg, t 42.15 min) and a mixture of compounds 1, 2 (31 mg, t
44.38-48.33 min). This mixture was further isolated by semi-preparative HPLC on
column #2, eluting with 60% MeOH , to obtain compounds 1 (8.5 mg) and 2 (11.6
mg), whose retention times were 68.62 and 71.05 min, respectively. The 40%
MeOH/CHCl eluate (1.7 g) was separated using MPLC system with an reverse-phase
3
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ODS column, eluting with MeOH/H
2
O (40%, 50%, 60%, 70%, 100%, each 500 mL),
to afford 5 fractions, of which Fraction 3 (35 mg) was further isolated by
semi-preparative HPLC on column #1, eluting with 65% MeOH65:35at the flow of 2
min/ml, to give compounds 4 (8.1 mg, t 37.45 min) and 5 (12 mg, t 40.32 min).
R
R
2
5
Compound 1: White amorphous powder; [α] -29.5 (c=0.12, MeOH); IR (KBr) νmax
:
D
-1
1
3455, 2941, 1761, 1645, 1462, 1047, 870 cm ; H-NMR (C D N, 500MHz) and
5
5
1
3
C-NMR (C D N, 125 MHz) spectroscopic data see table 1; HR-ESI-MS (negative
5
5
-
ion mode) m/z: 923.4646 [M-H] , ( C47
71
H O18; 923.4640).
2
5
Compound 2: White amorphous powder; [α] -25.4 (c=0.11, MeOH); IR(KBr) νmax
:
D
-1 1
3452, 2945, 2818, 2715, 1748, 1728, 1643, 1458, 1051, 883 cm ; H-NMR (C D N,
5
5
13
500MHz) and C-NMR (C
5
D
5
N, 125MHz) spectroscopic data see table 1 ;
-
HR-ESI-MS (negative ion mode) m/z: 981.4702 M-H] , ([C49
H
73
O20; 981.4695).
2
5
Compound 3: White amorphous powder; [α] -21.0(c=0.10, MeOH); IR(KBr)
D
-1 1
ν
5 5
max:3398, 2940, 2820, 2716, 1730, 1660, 1458, 1045, 887 cm ; H-NMR (C D N,
1
3
500MHz) and C-NMR (C
5
D
5
N, 125MHz) spectroscopic data see table 1;
+
HR-ESI-MS (positive ion mode) m/z:969.5059 [M+H] (C49
H
77
O
19; 969.5059).
2
5
Compound 4: White amorphous powder; [α] -23.2 (c=0.11, MeOH); IR(KBr) νmax
:
D
-1
1
13
3380, 2940, 1658, 1465, 1052, 884 cm ; H-NMR (C
(C N, 125 MHz) spectroscopic data see table 1; HR-ESI-MS (negative ion mode)
m/z: 971.4815 [M -H] , (C48
Compound 5: White amorphous powder; [α] -22.1(c=0.10, MeOH); IR(KBr) νmax
5 5
D N, 500MHz) and C-NMR
5
D
5
-
75
H O20; 971.4852).
2
5
:
D
-1
1
13
3414, 2942, 1663, 1447, 1039, 887 cm ; H-NMR (C
5 5
D N, 500MHz) and C-NMR
(C N, 125MHz) spectroscopic data see table 1; HR-ESI-MS (negative ion mode)
5
D
5
-
m/z: 957.5027 [M-H] , (C H O ; 957.5059).
48
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Acid hydrolysis and sugar analysis of five new triterpenoid saponins (1-5). The
procedure of the acid hydrolysis and the sugar analysis of five new triterpenoid
23
saponins (1-5) was performed according to the published literature. The detailed
2
3
process of acid hydrolysis and GC conditions were same as the reported. In the
study, the GC retention times of standard monosaccharides, i.e. D-glucuronic acid,
D-glucuronic acid methyl esterÈD-galactose, D-xylose, and L-arabinose, were 19.58,
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24.35, 25.55, 31.98, and 33.00 min, respectively.
MTT Assay for Cell Viability. MTT (Sigma-Aldrich, St. Louis, MO) assay was
employed to detect the cytotoxicity of compounds 1-10. Briefly, RAW264.7
macrophage cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China)
5
were planted into 96-well plates at a density of 10 cells/well, and subsequently
treated with the isolated saponins for 24 h, followed by MTT treatment (0.5 mg/Ml)
for 3 h at 37 °C. The UV absorbance values were detected at 570 nm.
Determination of Nitric Oxide Production by Griess Assay. The inhibitory
activities of compounds 1-10 on the NO production in LPS-induced RAW264.7
24
mouse macrophages were evaluated using the previously reported method. Briefly,
5
RAW264.7 cells were planted into 24-well plate at the density of 5 × 10 then
incubated with or without 1 µg/mL of lipopolysaccharide (LPS) at dosages of various
indicated compounds for 24 h. Nitrite levels (NO) in culture media were determined
by Griess assay (Beyotime Institute of Biotechnology, Haimen, China).
Statistical analysis. All data were presented as means ± SD. For analysis, one-way
analysis of variance (one-way-ANOVA) of GraphPad Prism 6.0 software was
employed to evaluate the significance (pØ0.05) of the intergroup.
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RESULTS AND DISCUSSION
Sequentially using D101 resin and ODS column chromatography, the MeOH extract
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was separated to afford five new compounds (1-5) and five known ones, i.e.,
oxytrogenin
3β-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-β-D-glucuronopyrano
25
26
27
side, 6,
soyasaponin Bh, 7,
soyasaponin Bb, 8,
complogenin
3β-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyran
2
8
29
oside, 9, dehydresoyasaponin I, 10 (Figure 1).
72
Compound 1 was white amorphous powder with formula as C47H O18 according to
-
-1
its HR-ESI-MS [M-H] data. The IR data at 1760 and 1640 cm indicated the
existence of γ-lactone and olefin group of compound 1, and an oligoglycoside
-1
1
structure was inferred by 3453 and 1047 cm . From the H-NMR spectrum, six
tertiary methyl groups (δ 1.39, 3H, s; 1.21, 3H, s; δ 1.19, 3H, s; 0.97, 3H, s; δ 0.80,
3H, s; 0.67 3H, s) were labeled as methyl C 23, 27, 29, 28, 26, and 25, respectively. In
addition, the character proton signals of an olefinic proton (δ 5.10 1H, m) and a
hydroxymethylene group (δ 4.25 1H, m; δ 3.27 1H, d, J = 11.5 Hz), as well as two
H
H
H
H
H
H
oxygenated-methine groups (δ 3.371H, J = 12.0, 4.5 Hz) and ( δ 4.19 1H, J = 11.5,
H
H
1
5.5 Hz), were observed in the H-NMR spectrum. A total of 47 carbon resonances
13
were appeared in the C-NMR spectrum, of which 30 were affiliated to a triterpene
skeleton and the remaining 17 to three sugar moieties. Three sugar residues were
confirmed by the three anomeric proton signals (δ 4.93, 1H, d, J = 8.0 Hz, ), (δH
H
5.51,1H, d, J = 7.0 Hz,), and (δ 6.14 1H, brs), which were further identified as H-1 of
H
glucuronic acid, xylose, and rhamnose, respectively. In the HSQC spectrum (Table 1),
C
they showed the correlations with the carbons at δ 105.6 (C-1 of glucuronic acid),
102.0 (C-1 of xylose), and 102.5 (C-1 of rhamnose), respectively. The types of sugar
residues of compound 1 were identified as L-rhamnose, D-xylose, and D-glucuronic
acid using the acid hydrolysis assay.
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ꢉꢆ
ꢉꢇ
ꢉꢈ
ꢀꢉ
ꢀꢀ
ꢀꢁ
ꢀꢂ
ꢀꢃ
ꢀꢄ
ꢀꢅ
ꢀꢆ
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ꢀꢈ
ꢁꢉ
ꢁꢀ
ꢁꢁ
ꢁꢂ
ꢁꢃ
ꢁꢄ
H
As shown in Figure 2, the HMBC correlations between the proton at δ 4.93 (H-1 of
C
glucuronic acid) and the carbon at δ 91.1 (C-3 of aglycone) displayed that the
glucuronic acid group was linked to C-3 of the aglycone. The HMBC correlations
between the proton at δ 5.51 (H-1 of xylose) and the carbon at δ 78.2 (C-2 of
H
C
glucuronic acid), between the proton at δ 6.14 (H-1 of rhamnose) and the carbon at
H
δ
C
77.7 (C-2 of xylose) indicated the linkage type of those sugar moieties of
compound as
1
α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyl-(1→2)-β-D-glucuronopyranosyl. In
terms of their JH-1, H-2 coupling constants, the β-anomeric configurations of the
glucuronopyranosyl and xylopyranosyl were determined, and the α-anomeric
configuration of rhamnopyranosyl unit was elucidated by the NOESY correlation
between the protons at δ
H
6.14 (H-1 of rhamnose) and δ
H
1.74 (H-6 of rhamnose)
1
13
30
(Figure 2). Comparing to the H- and C-NMR spectrum data of albiziasaponin A,
a triterpenoid glycoside isolated from the stems of Albizia myriophylla, compound 1
showed similarity to albiziasaponin A except for the existence of extra signals of one
xylopyranosyl group in replacement of those of the glucuronopyranosyl group located
at C-2 of glucuronopyranosyl methyl ester in the structure of albiziasaponin A. Thus,
compound 1 was identified as 3β,24-dihydroxy-22β,30-epoxy-30-oxoolean-12-en
3-O-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyl-(1→2)-β-D-glucuronopyranosid
e.
Compound 2, as white amorphous powder, has a molecular formula of C49
H
74
O
20
-
-1
according to its [M-H] data
.
In terms of the IR data, the hydroxyl group (3452 cm ),
-1
-1
γ-lactone group (1748 cm ), and formyl group (2818, 2715, 1728 cm ) were existed
13
in compound 2. The C-NMR spectrum data exhibited 48 carbon peaks, of which 18
were affiliated to three sugar moieties and the remaining 30 to a triterpene skeleton.
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ꢄꢉ
The aglycone NMR data of compound 2 were identical to those of compound 1. As
shown in Table 1, the three protons (δ
H
4.93,1H, d, J = 8.0 Hz; 5.68 1H, d, J = 8.0 Hz;
105.5, 101.7, 102.4 ),
6.16 1H, brs, ) were correlated with three carbons (δ
C
respectively, indicating the presence of glucuronic acid, galactose, and rhamnose
moieties. The types of sugar in compound 2 were identified as L-rhamnose,
D-galactose, and D-glucuronic acid by the acid hydrolysis assay. The NMR data of
the glycosidic chain of compound 2 were similar to those of soyasaponin Bh
(compound 7), with exception of an additional formyl group. This additional formyl
group was assigned to be located at C-3 of galactopyranosyl group, according to the
HMBC correlations between the carbon at δ 77.6(C-3 of galactose) and the proton at
C
δ
H
C
8.59 (H-1 of formyl group), the carbon at δ 164.2 (C-1 of formyl group) and the
proton at δ 4.57 (H-3 of galactose). Thus, compound 2 was identified as
H
3β,24-dihydroxy-22β,30-epoxy-30-oxoolean-12-en
3-O-α-L-rhamnopyranosyl-(1→2)-β-D-(3′′-O-ꢊꢋꢌꢍꢎꢏ)-galactopyranosyl-(1→2)-β-D-glu
curonopyranoside.
Compound 3, isolated as white amorphous powder, was deduced by HR-ESI-MS data
1
to possess a molecular formula of C49
data, seven tertiary methyl groups (δ
0.96, 3H, s; 0.84 3H, s; 0.69 3H, s), an olefinic proton (δ
hydroxymethylene group (δ 4.27, 3.24, 2H, m), and a methine group (δ 3.38, 1H, m)
H
76
O
19. According to the H-NMR spectrum
H
1.40, 3H, s; 1.28, 3H, s; 1.15,3H, s; 0.96, 3H, s;
5.24, 1H, m), a
H
H
H
were observed. In addition, as shown in HSQC spectrum, three anomeric protons (δ
4.93, 1H, d, J = 8.0 Hz; 5.69, 1H, d, J = 8.0 Hz;δ 6.18, 1H, brs) showed correlations
with three carbons at δ 106.1, 102.4, 103.0, respectively, indicating the existence of
H
C
three sugar units in compound 3. The sugar residues of 3 were identified as
L-rhamnose, D-galactose, and D-glucuronic acid using the acid hydrolysis assay. The
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ꢆꢂ
ꢆꢃ
ꢆꢄ
H- and C-NMR data of 3 were identical to those of dehydrosoyasaponin I
3
1
(compound 10),
except for the present of an additional formyl group in 3. The
78.4 suggested that the
downfield shift of the C-3 signal of galactose (+1.8 ppm) at δ
C
additional formyl group was linked to C-3 of the galactopyranosyl group, which was
further confirmed by the HMBC correlations of C-3 of galactose (δ 78.4) to H-1 of
C
formyl group (δ
H
8.59), and C-1 of formyl group (δ
compound
C
164.8) to H-3 of galactose (δ
H
4.47). Thus,
3
was identified as
3-O-α-L-rhamnopyranosyl-(1→2)-β-D-(3′′-O-formyl)-galactopyranosyl-(1→2)-β-D-gl
ucuronopyranoside.
Compound 4, as white amorphous powder, was of a molecular formula of C H O
76 20
48
-
1
according to the [M-H] data. In the H-NMR spectrum, the signals of six tertiary
methyl groups at (δ 1.54, 3H, s; 1.36 3H, s; 1.36, 3H, s; 1.00, 3H, s; 0.92, 3H, s; 0.64,
3H, s), a hydroxymethylene group (δ 4.23, 1H, m; 3.21, 1H, d, J = 11.5 Hz), an
olefinic proton (δ 5.52, 1H, brs), as well as two oxygenated-methine groups (δH
H
H
H
13
3.37,1H, J = 11.5, 3.5 Hz; 4.30, 1H, J = 10.5, 3.5 Hz) were observed. The C-NMR
spectrum exhibited 48 carbon signals, of which 18 were attributed to three sugar
moieties and the remaining 30 to a triterpene skeleton. In addition, three anomeric
H H H
proton signals (δ 4.93, 1H, d, J = 8.0 Hz; δ 5.72, 1H, d, J = 8.0 Hz; δ 6.22 1H, brs)
(Table 1) showed the correlations with three carbons (δC 105.1, 101.5, 102.1,
respectively) in the HSQC spectrum. The types of sugar residues of 4 were identified
as L-rhamnose, D-galactose, and D-glucuronic acid using the acid hydrolysis assay.
1
13
32
The H- and C-NMR data of 4 were similar to those of sophoraflavoside II except
for the significant downfield shifts (+101.0 ppm) of C-18 signal at δ 145.7 and C-19
C
(+82.2 ppm) at δ 123.7, accompanied with the significant upfield shifts of C-12
C
(-100.4 ppm) signal at δ 22.7 and C-13 (-105.4 ppm) at δ 38.9, on account of the
C
C
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ꢈꢄ
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ꢈꢆ
ꢈꢇ
ꢈꢈ
movement of the double bond from C-12/C-13 in sophoraflavoside II to C-18/C-19 in
compound 4. The assignment of the double bond location at C-18/C-19 was also
H C
confirmed by the HMBC correlations between H-19 at δ 5.52 with C-29 at δ 179.2
and C-30 at δ 28.3. Thus, compound 4 was identified as 3β,22β,24-trihydroxy
C
oxyolean-18(19)-ene-29-acid
3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyrano
side.
Compound 5 was isolated as white amorphous powder with molecular formula of
-
1
C H O based on its HR-ESI-MS [M-H] data. In the H-NMR, the signals of six
48
78 19
tertiary methyl groups (δ 1.39, 3H, s; 1.30, 3H, s; 1.03, 3H, s; 0.97 3H, s; 1.00, 3H, s;
H
0.67, 3H, s), a doublet methyl (δ
H
1.34, 3H, d, J = 10.0 Hz), and a methine (δ
H
3.28,
5.01,
1H, dd, J = 11.5, 4.5 Hz) were observed. The three anomeric proton signals (δ
H
1H, d, J = 8.0 Hz; 5.80, 1H, d, J = 8.5 Hz; 6.35, 1H, brs) showed their HSQC (Table 1)
correlations with three carbons at δ 105.1, 101.8, and 101.8, respectively, indicating
C
the existence of three sugar moieties. The sugar residues of compound 5 were
identified as L-rhamnose, D-galactose, and D-glucuronic acid, using the acid
1
13
hydrolysis assay. The H- and C-NMR data of the aglycone of compound 5 was
33
similar to those of punicanolic acid except for the downfield shift of C-3 (+11.6 ppm)
signal at δ 89.8, due to the glycosidation shift of the additional glycosidic chain
C
linked to C-3 of the aglycone in compound 5. The location of the glycosidic chain was
also deduced from the HMBC correlations between the proton at δ
glucuronic acid) and the car δ 89.8 (C-3 of aglycone). The NMR data of the
glycosidic chain of compound 5 were same as those of compound 4, suggesting the
linkage of the glycosidic chain was
H
5.01 (H-1 of
C
ꢂꢉꢉ
α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl -(1→2)-β-D-glucuronopyranosyl.
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ꢂꢉꢃ
ꢂꢉꢄ
ꢂꢉꢅ
ꢂꢉꢆ
ꢂꢉꢇ
ꢂꢉꢈ
ꢂꢀꢉ
ꢂꢀꢀ
ꢂꢀꢁ
ꢂꢀꢂ
ꢂꢀꢃ
ꢂꢀꢄ
ꢂꢀꢅ
ꢂꢀꢆ
ꢂꢀꢇ
ꢂꢀꢈ
ꢂꢁꢉ
ꢂꢁꢀ
ꢂꢁꢁ
ꢂꢁꢂ
ꢂꢁꢃ
ꢂꢁꢄ
Therefore,
compound
5
was
identify
as
punicanolic
acid
3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyrano
side.
Anti-inflammatory Activities Soyasaponins isolated from soybean were reported to
have a significant therapeutic effect on many inflammation-related diseases, such as
2
2
33
34
acute lung injury, hepatitis and diabetic nephropathy. Until now, only crude
extract of soyasaponins and few soyasaponins were confirmed to contribute to
35
anti-inflammatory properties.
In this study, anti-inflammatory activities of
compounds 1-10 were investigated by the assay of NO release using LPS-stimulated
RAW264.7 cells. As shown in Table 2, no cytotoxicityy was observed in all treated
RAW264.7 cells. Compounds 1, 2 and 5 displayed moderate anti-inflammatory
activities, with IC50 values of 18.8, 16.1, and 13.2 µM, respectively, comparing with
those of hexadecadrol (IC50=0.41 µM) which was used as the positive control. The
35
activities of compounds 3, 7, 8, 9 and 10 were weak. Compounds 4 and 6 showed
no anti-inflammatory activity. This could be the result of the presence of carboxylic
3
6, 37
acid in ꢐꢑꢒ structure, compared with other active soyasaponins,
but further studies
are needed for confirmation. Nevertheless, the findings from this study provides
evidences that soyasaponins could possess anti-inflammatory activity but with lower
cytotoxicity in macrophage-mediated inflammatory responses.
Funding
This work was financially supported by the Science and Technology Project of
Sichuan Province (2017NZ0006).
Notes
No competing conflict of interest is existed among all authors.
ACKNOWLEDGMENTS
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ꢂꢂꢁ
ꢂꢂꢂ
ꢂꢂꢃ
ꢂꢂꢄ
ꢂꢂꢅ
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ꢂꢂꢇ
ꢂꢂꢈ
ꢂꢃꢉ
ꢂꢃꢀ
ꢂꢃꢁ
ꢂꢃꢂ
ꢂꢃꢃ
ꢂꢃꢄ
ꢂꢃꢅ
ꢂꢃꢆ
ꢂꢃꢇ
ꢂꢃꢈ
ꢂꢄꢉ
The authors sincerely appreciate Dr. Jon Parcher at University of Mississippi for
reviewing the manuscript and providing the valuable suggestions.
Supporting Information Available: The spectroscopic data compounds 1-5 is
available on the websitehttp://pubs.acs.org.
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soyasaponin Bb retards disease progression in a murine model of polycystic kidney
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ꢃꢀꢉ
ꢃꢀꢀ
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ꢃꢁꢁ
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ꢃꢁꢄ
pseudo-acacia L. II. Chem. Pharm. Bull (Tokyo). 1993, 41, 553-556.
(26) Ali, Z.; Khan, S. I.; Khan, I. A.. Soyasaponin Bh, a triterpene saponin containing
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(27) Zha, L. Y.; Mao, L. M.; Lu, X. C.; Deng, H.; Ye, J. F.; Chu, X. W.; Sun, S. X.;
Luo, H. J.. Anti-inflammatory effect of soyasaponins through suppressing nitric oxide
production in LPS-stimulated RAW 264.7 cells by attenuation of NF-κB-mediated
nitric oxide synthase expression. Bioorg. Med. Chem. Lett. 2011, 21, 2415-2418.
(28) Cui, B.; Inoue, J.; Takeshita, T.; Kinjo, J.; Nohara, T.. Triterpene glycosides from
the seeds of Astragalus sinicus L. Chem. Pharm. Bull (Tokyo). 1992, 40, 3330-3333.
(29) Miyao, H.; Sakai, Y.; Takeshita T.; Kinjo J.; Nohara T.. Triterpene saponins from
Abrus cantoniensis (Leguminosae). I. Isolatioin and characterization of four new
saponins and a new sapogenol. Chem. Pharm. Bull (Tokyo). 1996, 44, 1222-1227.
(30) Yoshikawa, M.; Morikawa, T.; Nakano, K.; Pongpiriyadacha, Y.; Murakami, T.;
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ꢃꢁꢅ
ꢃꢁꢆ
ꢃꢁꢇ
ꢃꢁꢈ
ꢃꢂꢉ
ꢃꢂꢀ
ꢃꢂꢁ
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expression of IL-6 and TNF-α on diabetic rats serum and myocardial. Chin. J.
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soyasaponins and soyasapogenols. J. Agric. Food Chem., 2014, 62 , 8247–8255
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FIGURE CAPTIONS.
Figure 1 Structures of the compounds 1-10.
Figure 2 Key HMBC and NOESY correlations for 1-5.
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Figure 1
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1
2
O
HOOC
O
HO
HO
O
H
HO
OH
OH
O
O
O
C
H
O
O
H
O
H3C
HO
HO
3
OH
4
H
OH
H
H
COOH
HOOC
O
HO
HO
O
H
OH
OH
O
O
HO
O
H
O
H3C
HO
HO
OH
5
Figure 2
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Journal of Agricultural and Food Chemistry
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5 5
Table 1. NMR Spectroscopic Data for Compounds 1-5 (in C D N)
1
2
3
4
5
No.
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
1
2
3
4
5
6
7
8
9
38.8
26.8
91.1
44.1
56.2
18.7
33.5
43.0
47.8
36.6
25.3
1.32m ,0.78m
2.19m, 1.87m
38.6
26.5
1.30m ,0.77m
2.14m, 1.80m
3.36m
39.2
26.2
91.8
44.5
56.7
17.4
34.8
40.5
48.5
37.1
23.9
124.7
142.5
42.7
27.3
28.0
47.3
48.5
47.3
34.8
51.6
216.4
23.6
1.38m,0.85m 38.6
2.15m,1.82m 26.5
1.47m,0.75m
2.20m,1.85m
38.6
26.4
1.48m,0.76m
2.18m,1.80m
3.28dd(11.5,4.5)
3.37dd(12.0,4.5) 91.2
43.9
3.38m
91.0
43.8
56.0
3.37dd(11.5,3.5) 89.8
39.5
0.83m
56.1
18.5
33.3
42.9
47.7
36.5
24.9
125.1
140.9
39.7
25.2
26.6
36.1
45.2
42.7
42.4
38.5
0.83m
0.85m
0.73m
55.6
18.2
34.7
41.3
50.0
36.6
21.7
29.5
40.6
42.8
29.6
35.8
51.1
47.8
39.7
72.2
37.2
33.6
28.0
0.68m
1.32m, 1.24m
1.44m, 1.26m
1.54m,1.22m
1.43m, 1.27m
1.44m,1.24m 18.2
1.43m,1.28m 34.7
40.6
1.48m,1.20m
1.39m,1.21m
1.34m,1.23m
1.42m,1.25m
1.52m
1.52m
1.28m
51.0
36.5
1.22m
1.24m
1
0
11
1.66m,1.21m
1.67m,1.20m
5.10brs
1.51m,1.21m 21.4
1.43m,1.16m
1.82m,1.13m
2.41m
1.39m,1.13m
1.31m,1.17m
2.69m
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
125.2 5.10brs
141.0
5.24brs
27.4
38.9
43.2
26.7
39.8
25.1
26.6
36.2
45.3
42.8
42.5
38.6
84.7
23.1
1.66m, 1.21m
1.67m,1.21m
1.80m, 1.06m
1.68,1.36m
1.63m,1.32m
1.68m,0.96m
1.95m,1.23m
2.32m,1.55m
1.80m, 1.02m
2.12m,1.15m 28.3
39.9
2.11m
2.11m
2.35m
145.8
2.42m
1.79m,1.50m
1.79m,1.50m
2.02m,1.16m 123.7
5.52s
2.43d(3.5)
45.0
2.57m,2.14m 38.4
75.7
1.95m, 2.25m
1.96m, 2.25m
4.2m
2.92m,1.90m
4.3dd(10.5,3.5)
1.36s
2.05m,1.81m
2.30m,1.97m
1.30s
4.19dd(11.5,5.5) 84.6
1.39s
23.0
1.39s
1.40s
22.7
4.25m
4.25m
3.28d(11.0)
0.66s
4.27m
3.24m
0.69s
4.23m
6
3.6
63.6
64.2
63.3
16.1
1.03s
3
.27d(11.5)
3.21d(11.5)
0.64s
2
2
2
2
2
3
5
6
7
8
9
0
15.9
16.9
25.1
23.7
20.8
180.5
0.67s
0.8s
15.8
16.8
25.0
23.6
20.7
180.5
105.5
78.3
77.8
73.8
77.6
172.5
16.5
17.4
26.2
21.7
32.6
26.2
106.1
79.0
77.0
74.4
78.3
173.0
16.7
15.9
14.9
18.7
179.2
28.3
105.1
78.2
76.5
73.7
77.4
170.2
16.4
16.4
14.9
179.0
18.8
30.6
0.67s
0.97s
1.00s
0.8s
0.96s
1.00s
1.21s
0.97s
1.19s
1.20s
1.28s
0.92s
0.97s
1.15s
1.36s
1.18s
0.96s
1.34d(10.0)
1.39s
0.84s
1.54s
GlcA-1
105.6 4.93d(8.0)
4.93d(8.0)
4.17m
4.93d(8.0)
4.35
4.93d(8.0)
105.1 5.01d(8.0)
b
b
2
78.2
76.8
74.1
77.7
172.8
4.53m
4.55m
78.3
77.1
73.4
78.5
172.4
4.48m
4.58m
4.40m
4.58m
b
b
b
b
3
4.41m
4.44m
4.23m
4.05m
4.42m
4.53m
b
4
4.42m
4.57m
4.40m
b
b
b
5
4.44m
4.60m
4.50m
6
Xyl-1
102.0 5.51d(7.0)
2
3
77.7
75.8
4.58m
4.00
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b
4
70.7
67.1
4.00m
5
4.17m,3.57m
Gal-1
101.7
77.7
77.6
71.2
76.4
61.6
164.2
102.4
72.4
72.6
74.3
69.4
19.0
5.68d(8.0)
102.4
77.3
78.4
71.7
77.2
5.69d(8.0)
4.53m
101.5
77.3
76.5
70.9
76.4
61.4
5.72d(8.0)
101.8 5.80d(8.5)
b
b
2
4.45m
4.50m
78.4
79.2
72.6
77.5
63.2
4.26m
b
b
3
4.57m
4.47m
4.53m
4.19m
b
b
4
4.33m
4.35m
4.35m
4.02m
5
4.04m
3.90m
4.05m
3.81m
b
6
4.34m ,4.26m 62.2
4.36o,4.27m
8.59s
4.36m,4.28m
4.45,4.26m
Formyl
8.59s
164.8
103.0
73.0
73.3
75.0
70.0
Rha-1
102.5 6.14(brs)
6.16(brs)
4.69(brs)
4.63m
6.18(brs)
4.74(brs)
102.1
72.2
72.5
74.1
69.1
18.7
6.22(brs)
4.75(brs)
4.66m
101.8 6.35(brs)
2
3
4
5
6
72.5
72.8
74.5
69.5
19.1
4.73(brs)
4.64m
72.2
72.5
74.1
69.2
18.8
4.72(brs)
4.66m
b
4.63m
b
b
b
b
b
4.29m
4.27m
4.29m
4.29m
4.30m
b
4.92m
4.96m
4.93m
4.95m
5.01m
1.74d(6.0)
1.73d(6.5)
19.6
1.74d(7.0)
1.73d(6.0)
1.77d(6.0)
a 1
13
b
H at 500 MHz and C NMR at 125 MHz in DMSO-d
6
.. Signals were overlapped.
5
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Table 2. Anti-inflammatory Activities of compounds 1-10
Compou
nds
hexadeca
drol
1
2
3
4
5
6
7
8
9
10
IC50 for
inhibitio
n of NO
producti
on (µM)
cell
1
6
8.8±1. 16.1±1. 28.3±1.
51 97
13.2±1.
43
27.4±1. 26.7±1.
68 49
33.5±2. 28.6±2.
16 34
>50
>50
0.41±0.03
—
2
a
viability
9
8
7.6±8. 95.2±6. 93.4±9. 81.4±5.
96.8±6. 85.7±5.
92
96.1±9. 103.7±8
90.3±7. 93.8±7.
47 79
(
%
of
*
*
1
65
38
71
15
87
.62
cell
survival)
a
*
Cell viability was measured at appropriate IC50 values corresponding to the inhibition of NO, pØ0.05
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TOC graphic
8
5x47mm (300 x 300 DPI)
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