Triterpene Glycosides from the Seeds of Dolichos lablab

Article abstract of DOI:10.1248/cpb.c19-00155

Two new triterpene glycosides (1 and 2), together with nine known triterpene glycosides (3–11), were isolated from the seeds of Dolichos lablab (Leguminosae). The structures of the new compounds were determined by spectroscopic analysis, including two-dimensional NMR spectroscopy, and chromatographic analysis of the hydrolyzed products. The isolated compounds did not show cytotoxicity against HL-60 human leukemia cells and HepG2 human hepatoma cells at sample concentrations of 20μM.

Full text of DOI:10.1248/cpb.c19-00155

604
Chem. Pharm. Bull. 67, 604–608 (2019)
Vol. 67, No. 6
Note
Triterpene Glycosides from the Seeds of Dolichos lablab
Akihito Yokosuka,* Hiroki Takayama, and Yoshihiro Mimaki
Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences;
1432–1 Horinouchi, Hachioji, Tokyo 192–0392, Japan.
Received February 15, 2019; accepted March 14, 2019
Two new triterpene glycosides (1 and 2), together with nine known triterpene glycosides (3–11), were
isolated from the seeds of Dolichos lablab (Leguminosae). The structures of the new compounds were de-
termined by spectroscopic analysis, including two-dimensional NMR spectroscopy, and chromatographic
analysis of the hydrolyzed products. The isolated compounds did not show cytotoxicity against HL-60 human
leukemia cells and HepG2 human hepatoma cells at sample concentrations of 20µM.
Key words triterpene glycoside; Dolichos lablab; Leguminosae; cytotoxic activity
bearing methine carbons at δC 91.8 and 69.5, an oxygen-
Introduction
Dolichi Semen is prepared from the seeds of Dolichos bearing methylene carbon at δC 64.4, a set of olefinic carbons
lablab L., which belongs to the family Leguminosae (Faba- at δC 143.4 and 124.7, and an aldehyde carbon at δC 210.0. Acid
ceae) and is widely distributed in the tropical and subtropi- hydrolysis of 1 with 1M HCl in dioxane–H2O (1:1) yielded D-
cal countries of the world with the majority found in South galactose and D-glucuronic acid as the carbohydrate moieties,
China.1) This crude drug is listed in the 17th edition of the while the aglycone moiety decomposed under acidic condi-
Japanese Pharmacopoeia and has been used for treatment of tions. Identification of these sugars, including their absolute
indigestion, nausea, and diarrhea in Asian countries.2) Several configurations, was carried out by direct HPLC analysis of the
oleanane-type triterpene saponins, named lablabosides A–F,3,4) hydrolysate, which was performed on an amino-propyl bonded
flavonol glycosides,5) and a brassinosteroid6) have been isolat- silica gel column for D-galactose and on a sulfonated polysty-
ed from D. lablab seeds. As part of our ongoing investigations rene ion-exclusion column for D-glucuronic acid. These data
of traditional medicines, a phytochemical investigation was suggest that 1 is a triterpene diglycoside. On comparison of
1
carried out on the seeds of D. lablab. Two new triterpene gly- the H- and 13C-NMR spectral signals for the aglycone moiety
1
cosides (1 and 2) and nine known triterpene glycosides (3–11) of 1, which were assigned by analysis of the H–1H shift cor-
1
were isolated. In this paper, we describe the structural eluci- relation spectroscopy (COSY), the H-detected heteronuclear
1
dation of the two new triterpene glycosides (1 and 2) based single-quantum coherence (HSQC), and the H-detected het-
on spectroscopic analysis, including two-dimensional NMR eronuclear multiple-bond connectivity (HMBC), with those
spectroscopy, and chromatographic analysis of the hydrolyzed of lablaboside B (8), the signals due to the ring A–D portions
products. The isolated compounds (1–11) were evaluated for (C-1–C-16 and C-23–C-27) were observed at almost the same
cytotoxicity against HL-60 human leukemia cells and HepG2 positions between the two compounds, indicating the presence
human hepatoma cells.
of the C-3β oxygen atom bearing the sugar residue, C-12/C-13
double bond, and C-24 hydroxy group. The 4β (4S) configu-
ration was ascertained by nuclear Overhauser effect (NOE)
Results and Discussion
The MeOH extract of the seeds of D. lablab (5.0kg) was correlations between H-3 and Me-23/H-5, and between H-24a
repeatedly subjected to column chromatography, yielding and Me-25 in the phase-sensitive NOE spectroscopy spectrum
1
compounds 1–11 (Fig. 1).
of 1. The H- and 13C-NMR data for the ring E portion of 1
Compound 1 was obtained as an amorphous solid and had were different from those of 8, and suggested the presence of
a molecular formula of C42H66O15, as determined from the a hydroxy group (δH 3.79/δC 69.5) and an aldehyde group (δH
high resolution (HR)-electrospray ionization (ESI)-time of 9.55/δC 210.0). In the HMBC spectrum, the aldehyde proton
flight (TOF)-MS (m/z: 833.4263 [M+Na]+) and 13C-NMR (δH 9.55) showed long-range correlations with the methylene
data. The IR spectrum showed the absorptions for the hy- carbon at δC 16.3 (C-16), quaternary carbon at δC 55.9 (C-17),
droxy (3417cm−1) and carbonyl (1725cm−1) functionalities. and methine carbon at δC 42.3 (C-18). The hydroxy methine
1
The H-NMR spectrum of 1 contained signals for six methyl proton (δH 3.79) exhibited spin-coupling correlations with
groups derived from the triterpene skeleton at δH 1.22, 1.18, methylene protons at δH 1.41 and 1.39 (H2-21), and HMBC
0.99, 0.95, 0.86, and 0.72 (each s), an olefinic proton at δH 5.30 correlations with the quaternary carbon at δC 55.9 (C-17) and
(brs), an aldehyde proton at δH 9.55 (brs), and two anomeric aldehyde carbon (δC 210.0). These correlations allowed an al-
protons at δH 4.70 (d, J=7.7Hz) and 4.47 (d, J=7.3Hz). The dehyde group and a hydroxy group to be placed at C-17 and
13C-NMR spectrum contained two anomeric carbon signals of C-22, respectively (Fig. 2). The spin-coupling constants of
3
the sugar moiety at δC 104.9 and 104.8, and 30 carbon signals H-22 (3JH-22,H-21axial =11.5Hz, JH-22,H-21equatorial =5.9Hz), and
of the aglycone moiety including signals for six methyl car- NOE correlations between H-18 and H-22/Me-30, H-21α and
bons at δC 33.5, 26.2, 24.9, 23.0, 17.6, and 16.1, six quaternary Me-29, and between H-21β and Me-30 were consistent with
carbons at δC 55.9, 44.6, 43.1, 40.9, 37.5, and 32.3, two oxygen- the 22α (22S) configuration (Fig. 3). Combined analysis of
*To whom correspondence should be addressed. e-mail: yokosuka@toyaku.ac.jp
© 2019 The Pharmaceutical Society of Japan

Vol. 67, No. 6 (2019)
Chem. Pharm. Bull.
605
Fig. 1. Chemical Structures of 1–11
Fig. 3. Important NOE Correlations of 1
Compound 2 was found to have a molecular formula
of C48H76O19 by HR-ESI-TOF-MS analysis (m/z 979.4849
Fig. 2. Partial Structure of 1
Bold lines indicate the 1H–1H spin-couplings and arrows indicate 1H/13C long-
range correlations.
1
[M+Na]+). The H- and 13C-NMR spectra of 2 revealed that
the aglycone of 2 was the same as that of 1. The deduced
the H–1H COSY and HSQC spectra indicated that the digly- molecular formula of 2 was higher than that of 1 by C6H10O5,
1
1
coside moiety of 1 was composed of a C-2-substituted β-D- corresponding to one hexose unit, and the H-NMR spectrum
glucuronopyranosyl (4C1) unit (GlcUA) [δH 4.47 (d, J=7.3Hz); of 2 contained signals for three anomeric protons at δH 6.32
δC 104.9, 80.9, 78.5, 73.5, 76.8, and 176.0] and a terminal β-D- (d, J=1.6Hz), 4.87 (d, J=7.6Hz), and 4.44 (d, J=7.8Hz), and
galactopyranosyl (4C1) unit (Gal) [δH 4.70 (d, J=7.7Hz); δC the methyl group of a 6-deoxyhexose at δH 1.58 (d, J=6.4Hz).
104.8, 73.4, 75.2, 71.2, 76.9, and 62.7]. The anomeric configu- Acid hydrolysis of 2 yielded D-galactose, D-glucuronic acid,
1
rations of the GlcUA and Gal residues were determined as β and L-rhamnose. On comparison of the H- and 13C-NMR
3
by the large JH-1, H-2 values of their anomeric protons (7.3 and spectra of 2 with those of 1, a set of six additional signals
7.7Hz, respectively). In the HMBC spectrum of 1, long-range corresponding to a terminal α-L-rhamnopyranosyl unit (Rha)
correlations were observed between the anomeric proton (H-1) [δH 6.32 (1H, d, J=1.6Hz); δC 102.3, 72.3, 72.2, 74.3, 69.6,
of Gal at δH 4.70 and C-2 of GlcUA at δC 80.9, and between and 18.6] appeared, and the signal due to C-2 of the galac-
H-1 of GlcUA at δH 4.47 and C-3 of the aglycone at δC 91.8. tosyl moiety shifted downfield by 4.7ppm to be observed
Accordingly, the structure of 1 was established as 3β-[(2- at δC 78.1. The anomeric proton of Rha was equatorial, and
O-β-D-galactopyranosyl-β-D-glucuronopyranosyl)oxy]-22α,24- thus possessed an α-pyranoid anomeric form (1C4) due to
1
dihydroxyolean-12-en-28-al.
the large JC-1, H-1 value (174.2Hz).7) In the HMBC spectrum

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Chem. Pharm. Bull.
Vol. 67, No. 6 (2019)
of 2, long-range correlations were observed between the sulfate (Gibco, Grand Island, NY, U.S.A.), and phosphate
anomeric proton (H-1) of Rha at δH 6.32 and C-2 of Gal at buffered saline (PBS) (Wako Pure Chemical Industries, Ltd.,
δC 78.1, H-1 of Gal at δH 4.87 and C-2 of GlcUA at δC 77.3, Osaka, Japan). All other chemicals used were of biochemical
and between H-1 of GlcUA at δH 4.44 and C-3 of the agly- reagent grade.
cone at δC 92.3. Thus, the structure of 2 was assigned as
3β-[(2-O-α-L-rhamnopyranosyl-2-O-β-D-galactopyranosyl-β-D- lablab) were purchased from Tochimoto Tenkaido Co., Ltd,.
glucuronopyranosyl)oxy]-22α,24-dihydroxyolean-12-en-28-al.
(Osaka, Japan) in April 2012. A voucher specimen has been
Plant Material Dolichi Semen (dried seeds of Dolichos
Compounds 3–11 were identified by their physical and deposited in our laboratory (voucher no. DL-2012-001, De-
spectroscopic data as chikusetsusaponin IVa (3),8) gypsogenin partment of Medicinal Pharmacognosy).
3-O-β-D-glucuronopyranosyl-28-O-β-D-glucopyranoside
Extraction and Isolation Dolichi Semen (5.0kg) was
(4),9,10) quinoasaponin 9 (5),11) udosaponin B (6),12,13) lablabo- extracted with hot MeOH (20L). The MeOH extract was
side A (7),3) lablaboside B (8),3) lablaboside C (9),3) lablaboside concentrated under reduced pressure, the viscous concentrate
F (10),4) and lablaboside E (11),4) respectively. This is the first (195g) was subjected to a Diaion HP-20 column, and succes-
report concerning the isolation of 4–6 from D. lablab.
sively eluted with 30% MeOH, 50% MeOH, MeOH, EtOH,
The isolated compounds (1–11) were evaluated for their cy- and EtOAc (each 6L). The 50% MeOH eluted fraction (6.0g)
totoxic activity against HL-60 and HepG2 cells. They did not was separated by silica gel CC eluted with a stepwise gradient
exhibit cytotoxicity even at sample concentrations of 20µM mixture of CHCl3–MeOH–H2O (90:10:1, 40:10:1, 20:10:1,
(<50% growth inhibition), whereas cisplatin gave IC50 values and 10:10:1), and finally with MeOH alone, to produce 10
of 1.22 0.15 (HL-60) and 4.40 0.27µM (HepG2).
fractions (A–J). Fraction F was purified by ODS silica gel CC
In summary, 11 triterpene glycosides (1–11), including two eluted with MeOH–H2O (2:3 and 3:2) and MeCN–H2O (1:3
new compounds (1 and 2), were isolated from the seeds of D. and 1:2), and silica gel CC eluted with EtOAc–MeOH–H2O
lablab, and their cytotoxicity against HL-60 cells and HepG2 (40:10:1 and 20:10:1) to give 1 (5.2mg), 3 (15.3mg), and 4
cells was evaluated. The isolated compounds did not show (5.9mg). Fraction G was purified by ODS silica gel CC eluted
cytotoxicity against HL-60 cells and HepG2 cells. Further with MeCN–H2O (2:7, 2:5, and 2:3) and MeOH–H2O (2:3
biological investigation of the isolated compounds is currently and 3:2), and silica gel CC eluted with EtOAc–MeOH–H2O
under way.
(70:40:1) to give 4 (7.0mg) and 5 (7.7mg). Fraction H was
purified by ODS silica gel CC eluted with MeCN–H2O (2:7,
2:5, and 1:2) to give 2 (9.7mg) and 6 (3.8mg). Fraction I
Experimental
Optical rotations were measured by using a JASCO P-1030 was purified by ODS silica gel CC eluted with MeCN–H2O
(Tokyo, Japan) automatic digital polarimeter. IR spectra (2:7 and 1:3) and MeOH–H2O (1:1) to give 7 (9.0mg), 8
were recorded on a JASCO FT-IR 620 spectrophotometer. (4.7mg), 9 (5.0mg), and 11 (9.0mg). Fraction J was separated
NMR spectral data were obtained on a DRX-600 (600MHz by an ODS silica gel column eluted with MeOH–H2O (1:1) to
for 1H-NMR, Bruker, Karlsruhe, Germany) and DRX-500 yield subfractions J-1–J-9. Fraction J-5 was purified by ODS
1
spectrometer (500MHz for H-NMR, Bruker) using standard silica gel CC eluted with MeCN–H2O (2:7), and silica gel CC
Bruker pulse programs at 300K. Chemical shifts were pre- eluted with EtOAc–MeOH–H2O (10:10:1) to give 11 (9.8mg).
sented as δ values with reference to tetramethylsilane (TMS) Fraction J-6 was purified by silica gel CC eluted with EtOAc–
as an internal standard. ESI-TOF-MS data were recorded on MeOH–H2O (20:10:1, 30:20:1, 10:10:1, and 10:20:1), and
an LCT mass spectrometer (Waters-Micromass, Manchester, ODS silica gel CC eluted with MeOH–H2O (1:1) to give 10
U.K.). Diaion HP-20 (Mitsubishi-Chemical, Tokyo, Japan), (4.8mg).
silica gel (Fuji-Silysia Chemical, Aichi, Japan), and octa-
decylsilanized (ODS) silica gel (Nacalai Tesque, Kyoto, Japan)
Compound 1
Amorphous solid; [α]D25 +38.2 (c=0.10, MeOH); IR
were used for column chromatography (CC). TLC was per- νmax (film) cm−1: 3417 (OH), 2924 (CH), 1725 (C=O); H-
formed on precoated silica gel 60 F254 (0.25mm thick, Merck, (600MHz, CD3OD) and 13C-NMR (150MHz, CD3OD) spectra,
Darmstadt, Germany) and RP18 F254S plates (0.25mm thick, see Table 1; HR-ESI-TOF-MS m/z: 833.4263 [M+Na]+ (Calcd
Merck), and spots were visualized by spraying the plates for C42H66NaO15: 833.4299).
1
with 10% H2SO4 aqueous solution and heating. HPLC was
performed with a system consisting of a CCPM pump (Tosoh,
Compound 2
Amorphous solid; [α]D25 −5.3 (c=0.10, MeOH); IR
Tokyo, Japan), a Shodex OR-2 (Showa-Denko, Tokyo, Japan) νmax (film) cm−1: 3390 (OH), 2928 (CH), 1716 (C=O); H-
detector, and a Rheodyne injection port (Rohnert Park, CA, (600MHz, CD3OD) and 13C-NMR (150MHz, CD3OD) spectra,
U.S.A.). HL-60 cells (JCRB0085) and HepG2 cells (JCRB see Table 1; HR-ESI-TOF-MS m/z: 979.4849 [M+Na]+ (Calcd
1054) were obtained from the Japanese Collection of Research for C48H76NaO19: 979.4879).
1
Bioresources (JCRB) cell bank (Osaka, Japan). The following
Acid Hydrolysis of 1 and 2 Compounds 1 (2.5 mg) and
materials and reagents were used for the cell culture assays: a 2 (7.9 mg) were independently dissolved in 1M HCl (diox-
Spectra Classic microplate reader (Tecan, Salzburg, Austria), ane–H2O, 1:1, 2.0mL) and were heated at 95°C for 1h under
96-well flat-bottom plates (Iwaki Glass, Chiba, Japan), fetal an Ar atmosphere. After dilution of the reaction mixture with
bovine serum (FBS) (Nichirei Biosciences, Tokyo, Japan), H2O (5mL), it was extracted with EtOAc (10mL×3). The
0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) solu- H2O residue was neutralized by the addition of Ag2CO3. The
tion, RPMI 1640 medium, Dulbecco’s modified Eagle’s medi- mixture was filtered and then passed through a Sep-Pak C18
um (DMEM), cisplatin, and 3-(4,5-dimethylthiazol-2-yl)-2,5- cartridge (Waters, Milford, MA, U.S.A.) eluted with H2O
diphenyl-2H-tetrazolium bromide (MTT) (Sigma-Aldrich, St. (5mL×3) to give sugar fractions (1.0mg from 1, and 3.3mg
Louis, MO, U.S.A.), penicillin G sodium salt and streptomycin from 2). The sugar fractions were each analyzed by HPLC

Vol. 67, No. 6 (2019)
Chem. Pharm. Bull.
607
Table 1. 1H- and 13C-NMR Chemical Shift Assignments for 1 and 2 (δ, ppm: J, Hz)a)
1
2
Position
δH
δC
δH
δC
1 eq
ax
1.60 m
1.02 m
2.12 m
1.80 m
39.6
1.61 m
1.03 m
2.11 m
1.80 m
39.6
2 eq
ax
27.0
27.0
3
3.41 brd (9.6)
91.8
44.6
57.3
19.4
3.40 brd (9.5)
92.3
44.7
57.2
19.3
4
5
0.93 m
1.61 m
1.34 m
1.34 m
1.52 m
0.92 m
1.61 m
1.33 m
1.33 m
1.53 m
6 eq
ax
7 eq
ax
34.1
34.1
8
40.9
48.9
40.9
48.8
9
1.56 dd (10.3, 6.9)
1.56 dd (10.2, 7.1)
10
37.5
37.5
11 (2H)
12
1.89 m
24.7
1.88 m
24.7
5.30 brs
124.7
143.4
43.1
5.30 brs
124.7
143.4
43.1
13
14
15 eq
ax
1.78 m
27.0
1.78 m
27.0
1.14 m
1.14 m
16 eq
ax
2.04 brd (9.1)
1.77 m
16.3
2.04 brd (9.1)
1.77 m
16.1
17
55.9
42.3
46.1
55.9
42.3
46.1
18
2.64 dd (13.6, 4.6)
1.14 dd (13.6, 4.6)
1.77 dd (13.6, 13.6)
2.64 dd (13.6, 3.9)
1.14 dd (13.6, 3.9)
1.78 dd (13.6, 13.6)
19 eq
ax
20
32.3
43.1
32.3
43.1
21 eq
ax
1.41 m
1.40 dd (12.4, 5.9)
1.39 dd (12.4, 11.2)
3.79 dd (11.2, 5.9)
1.25 s
1.39 m
22
3.79 dd (11.5, 5.9)
1.22 s
69.5
23.0
64.4
69.5
23.4
64.3
23
24 a
b
4.11 d (11.5)
3.21 d (11.5)
0.86 s
4.13 d (11.8)
3.18 d (11.8)
0.85 s
25
16.1
17.6
26.2
210.0
33.5
24.9
104.9
80.9
78.5
73.5
76.8
176.0
104.8
73.4
75.2
71.2
76.9
62.7
16.1
17.6
26.2
210.1
33.5
24.9
105.5
77.3
78.4
74.1
76.9
176.6
102.1
78.1
76.3
71.6
76.3
62.2
26
0.72 s
0.71 s
27
1.18 s
1.18 s
28
9.55 s
9.56 s
29
0.95 s
0.95 s
30
0.99 s
0.99 s
GlcUA 1′
4.47 d (7.3)
3.61 dd (9.5, 7.3)
3.62 dd (9.5, 9.5)
3.47 dd (9.5, 9.5)
3.55 d (9.5)
4.44 d (7.8)
3.75 dd (9.3, 7.8)
3.60 dd (9.3, 9.3)
3.42 dd (9.3, 9.3)
3.53 d (9.3)
2′
3′
4′
5′
6′
Gal 1″
2″
4.70 d (7.7)
4.87 d (7.6)
3.54 dd (9.7, 7.7)
3.43 dd (9.7, 3.4)
3.76 brd (3.4)
3.50 m
3.64 dd (9.6, 7.6)
3.53 dd (9.6, 3.3)
3.71 brd (3.3)
3.48 m
3″
4″
5″
6″ a
3.79 dd (11.8, 1.7)
3.68 dd (11.8, 4.7)
3.75 dd (11.7, 1.5)
3.69 dd (11.7, 5.4)
6.32 d (1.6)
b
Rha 1‴
2‴
102.3
72.3
72.2
74.3
69.6
18.6
4.93 dd (3.3, 1.6)
4.75 dd (9.5, 3.3)
4.46 dd (9.5, 9.5)
4.66 m
3‴
4‴
5‴
6‴
1.58 d (6.4)
a) Spectra were measured in CD3OD.

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Chem. Pharm. Bull.
Vol. 67, No. 6 (2019)
under the following conditions: 1) column, Aminex HPX-87H
(7.8mm i.d. ×300mm, 5µm, Bio-Rad Laboratories, CA,
U.S.A.); solvent, 5 mM H2SO4 in H2O; flow rate, 0.6mL/min;
detection, tR (min) and optical rotation (OR). D-Glucuronic
acid was detected in the sugar fractions obtained from 1 and
2 (tR 8.23, positive optical rotation). 2) column, Capcell Pak
NH2 UG80 (4.6mm i.d. ×250mm, 5µm, Shiseido, Tokyo,
Japan); solvent, MeCN–H2O (17:3); flow rate, 1.0mL/min;
detection, tR (min) and OR. D-Galactose was detected in the
sugar fractions obtained from 1 and 2 (tR 10.58, positive opti-
cal rotation), and L-rhamnose in that from 2 (tR 6.58, negative
optical rotation).
Cell Culture and Assay for Cytotoxic Activity against
HL-60 and HepG2 Cells HL-60 cells (JCRB 0085) and
HepG2 cells (JCRB 1054) were obtained from the Japanese
Collection of Research Bioresources (Osaka, Japan). The
cells were cultured in RPMI 1640 medium (HL-60 cells) or
DMEM (HepG2 cells) containing heat-inactivated 10% (v/v)
FBS supplemented with L-glutamine, 100unit/mL penicillin G
sodium salt, and 100µg/mL streptomycin sulfate in a humidi-
fied incubator at 37°C with an atmosphere of 5% CO2. The
cells (HL-60: 4×104 cells/mL, HepG2: 4×104 cells/mL) were
treated with each compound for 72h continuously, followed by
the measurement of cell viability by using an MTT assay.14)
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Conflict of Interest The authors declare no conflict of
interest.