New antimicrobial and cytotoxic acylated triterpenoidal saponins from Gleditsia aquatica

Article abstract of DOI:10.1007/s00044-015-1345-5

Phytochemical investigation on the fruits of Gleditsia aquatica resulted in the isolation and identification of two new bisdesmosidic triterpenoidal saponins, aquaticasaponin A (1) and aquaticasaponin B (2) acylated with two and one monoterpenic acids, respectively, and one known cytokinin, aquaticine C (3). The structural elucidation of isolated metabolites was established on the basis of 1D, 2D NMR, and MS spectral analyses. The antimicrobial activity of the isolated compounds [1-3] was evaluated. Compound 1 exhibited the highest degree of activity against Syncephalastrum racemosum with an MIC value of 9.2 μM, whereas compound 2 exhibited the highest degree of activity against Escherichia coli with an MIC value of 67.3 μM. The isolated compounds also exhibited good cytotoxic activity against human breast cancer (MCF-7) and human colon cancer (HCT-116) cell lines with values of IC₅₀ from 0.5 to 1.0 μM. Compound 1 was found to be the most active against colon cancer HCT-116 cell line with IC₅₀ value of 0.5 μM.

Full text of DOI:10.1007/s00044-015-1345-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1345-5
ORIGINAL RESEARCH
New antimicrobial and cytotoxic acylated triterpenoidal saponins
from Gleditsia aquatica
Ehab A. Ragab
Received: 18 September 2014 / Accepted: 19 February 2015
Ó Springer Science+Business Media New York 2015
Abstract Phytochemical investigation on the fruits of
Gleditsia aquatica resulted in the isolation and identifica-
tion of two new bisdesmosidic triterpenoidal saponins,
aquaticasaponin A (1) and aquaticasaponin B (2) acylated
with two and one monoterpenic acids, respectively, and
one known cytokinin, aquaticine C (3). The structural
elucidation of isolated metabolites was established on the
basis of 1D, 2D NMR, and MS spectral analyses. The
antimicrobial activity of the isolated compounds [1–3] was
evaluated. Compound 1 exhibited the highest degree of
activity against Syncephalastrum racemosum with an MIC
value of 9.2 lM, whereas compound 2 exhibited the
highest degree of activity against Escherichia coli with an
MIC value of 67.3 lM. The isolated compounds also ex-
hibited good cytotoxic activity against human breast cancer
(MCF-7) and human colon cancer (HCT-116) cell lines
with values of IC50 from 0.5 to 1.0 lM. Compound 1 was
found to be the most active against colon cancer HCT-116
cell line with IC50 value of 0.5 lM.
Keywords Gleditsia aquatica ꢀ Fabaceae ꢀ
Acylated oleanane-type triterpenoidal saponins ꢀ
Antimicrobial activity ꢀ Cytotoxic activity
Introduction
Genus Gleditsia L. (Fabaceae) has long been known in tra-
ditional Chinese folk medicine and used for the treatment of
apoplexy, as an expectorant and as a pesticide (Zhong and
Dian, 1977). Saponins (Zang et al., 1999a, b, c, d) and cy-
tokinins (Hosny et al., 2009) have been reported from some
species of this genus. In the flora of Egypt, the genus Gled-
itsia is represented by three species: G. aquatica March., G.
caspia Desf, and G. triacanthos. L. Gleditsia aquatica
March. is a perennial shrub distributed throughout Egypt.
Two new acylated triterpenoidal saponins, aquaticosides A
and B, and two new cytokinins, aquaticine A and B were
isolated from the fruits of G. aquatica (Ragab et al., 2010).
Further, phytochemical study of the fruits of this plant has led
to the isolation of another two new saponins, termed
aquaticasaponin A (1) and aquaticasaponin B (2) and one
known cytokinin, named aquaticine C (3) (Fig. 1). This pa-
per deals with the elucidation of their structures by extensive
NMR studies, including DEPT, DQF-COSY, HMQC, and
HMBC experiments and the results of hydrolytic cleavage.
The two saponins consisting of the same aglycon and the
same sugar sequence were acylated with one or two
monoterpenic acid units at C-2 position or at C-2 and C-3
positions of the rhamnose moiety, which is attached to C-6
position of the glucose moiety, which is directly connected to
the C-28 carbonyl group of the aglycon. Antimicrobial and
cytotoxic activities of the isolated compounds (1–3) have
been studied and significant results were obtained.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-015-1345-5) contains supplementary
material, which is available to authorized users.
E. A. Ragab (&)
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar
University, Cairo 11371, Egypt
e-mail: ar_ehab@yahoo.com
123

Med Chem Res
O
Ara
C
OH
Glc-NAc
HO
HO
O
O
O
O
HO
HO
O
OH
NH
C
O
Glc
O
Rha'
O
CH3
H3C
HO
O
O
O
HO
HO
HO
HO
1
2
R1 = R2 =
R2O
OR1
O
Rha
H3C
O
Xyl
O
Xyl'
O
HO
HO
O
OH
O
HO
HO
OH
R1
=
R2 = H
OH
15
13
11
10
HN
14
12
6
N
5
4
N
8
Glc
2
Api
O
OH
O
O
N
6`
N
OH
1``
H
4``
5``
1`
HO
OH
HO
OH
OH
3
Fig. 1 Compounds 1–3
Materials and methods
General experimental procedures
equipped with an IBM Aspect-2000 processor and with
software VNMR version 4.1 or NUTS program for NMR.
HRESIMS spectra were measured using a Bruker Bioapex-
FTMS with electrospray ionization (ESI). ESIMS was
carried out on a TSQ700 triple quadruple instrument
(Finnegan, Santos, CA, USA) mass spectrometer. EIMS
was carried out on Scan EIMS-TIC, VG-ZAB-HF, X-mass
(158.64, 800.00) mass spectrometer (VG Analytical, Inc.).
Polyamide (ICN Biomedicals) and Si gel (Si gel 60, Mer-
ck) were used for open column chromatography. Flash
column liquid chromatography was performed using J.T.
Baker glassware with 40 lm Si gel (Baker) and Sepralyte
C18 (40 lm) as the stationary phase. TLC was carried out
on precoated silica gel 60 F254 (Merck) plates. Developed
chromatograms were visualized by spraying with 1 %
vanillin-H2SO4, followed by heating at 100 °C for 5 min or
spraying the developed plates with 2 % ninhydrin in
acetone.
UV spectra were determined with a Hitachi 340 spec-
trophotometer; IR spectra were carried out on a Nicolet 205
FT-IR spectrometer connected to a Hewlett–Packard Color
1
Pro. Plotter. The H- and 13C-NMR measurements were
obtained with a Bruker NM spectrometer operating at 600
1
and 400 MHz (for H) and 100 MHz (for 13C), a Bruker
ARX-500, and a Jeol JNM ECA 500 NMR spectrometer
1
operating at 500 MHz (for H) and 125 MHz (for 13C) in
DMSO-d6 or CD3OD solutions, and chemical shifts were
expressed in d (ppm) with reference to TMS, and coupling
constant (J) in Hertz. 13C multiplicities were determined by
the DEPT pulse sequence (135°). DQF-COSY, HMBC, and
HMQC NMR experiments were carried out using a Bruker
AMX-600 and a Bruker ARX-500 high field spectrometer
123

Med Chem Res
Plant material
respectively. The tubes were then inoculated with the test
organisms, grown in their suitable broth for tested patho-
genic bacteria (1 9 108 CFU/mL for bacteria and 1 9
104 spore/mL for fungi); each 0.5 mL received 100 lL of
the above inoculum and was incubated at 37 °C for 24 h for
bacteria and after 48 h of incubation at 28 °C for fungi. MIC
values were taken as the lowest compound concentration that
prevents visible bacterial growth. Each experiment was
made three times. The results are presented in (Table 4).
The fruits of G. aquatica March. were collected from
agricultural Museum-Dokki, Giza, Egypt in March 2001
and were kindly identified by Engineer Badeia Hassan
Aly Dewan, Consultant of Egyptian Flora, Agricultural
Museum, Dokki, Giza, Egypt, and by Mrs. Terase Labib,
Taxonomist of Orman Garden, Giza, Egypt. A voucher
specimen {G-01} has been deposited in the Pharma-
cognosy Department, Faculty of Pharmacy, Al-Azhar
University, Cairo, Egypt.
Cytotoxicity assays
Antimicrobial assays
The compounds (1–3) were tested for cytotoxicity against
two human tumor cell lines: breast cancer (MCF-7) and
colon cancer (HCT-116) cell lines. The cells were obtained
from the American Type Culture Collection (ATCC,
Rockville, MD, USA). The cells were grown on Roswell
Park Memorial Institute (RPMI) 1640 medium (Nissui
Pharm. Co., Ltd., Tokyo, Japan) supplemented with 10 %
inactivated fetal calf serum and 50 lg/mL gentamycin. The
cells were maintained at 37 °C in a humidified atmosphere
with 5 % CO2 and were sub-cultured 2–3 times a week.
The cytotoxic activity was determined by using cell via-
bility assay method as described previously (Mosmann,
1983). The experiments were performed in triplicates, and
the percentage of cell viability was calculated as the mean
absorbance of control cells/mean absorbance of treated
cells. Dose–response curves were prepared, and the IC50
value was determined. The results are presented in
(Table 5).
Antimicrobial activities of compounds 1–3 were investi-
gated in vitro against different bacteria and fungi. Two
standard strains of Gram-positive bacteria [Streptococcus
pneumonia (RCMB 010010) and Bacillus subtilis (RCMB
010067)] and two standard strains of Gram-negative bacteria
[Pseudomonas aeruginosa (RCMB 010043) and Escher-
ichia coli (RCMB 010052)] were used for antibacterial as-
say. Four clinical pathogenic fungi [Aspergillus fumigates
(RCMB 02568), Syncephalastrum racemosum (RCMB
05922), Geotricum candidum (RCMB 05097), and Candida
albicans (RCMB 05036)] were used for antifungal assay.
The microbial species are environmental and clinically
pathogenic microorganisms obtained from Regional Center
for Mycology and Biotechnology antimicrobial unit
(RCMB), Al-Azhar University. Antimicrobial tests were
carried out by the agar well diffusion method (Scott, 1989),
using 100 lL of suspension containing 1 9 108 colony
forming units (CFU)/mL for tested bacteria and 1 9 104
spore/mL fungi spread on nutrient agar and malt extract agar,
respectively. After the media had cooled and solidified, wells
(6 mm in diameter) were made in the solidified agar and
loaded with 100 lL of tested compound solution in 1 mL
dimethyl sulfoxide (DMSO) with concentrations of 13.5,
14.7, 48.7 lM for compounds 1–3, respectively. Negative
controls were prepared using DMSO employed for dissolv-
ing the tested compound, while ampicillin, gentamycin, and
amphotericin B were used as positive controls for Gram-
positive bacteria, Gram-negative bacteria, and fungi, re-
spectively. The inoculated plates were then incubated for
24 h at 37 °C for bacteria and 48 h at 28 °C for fungi, and the
diameter of any resulting zones of inhibition of growth was
measured in millimeter (mm). Each experiment was per-
formed in triplicates, and the data were expressed as
mean SD. The minimal inhibitory concentrations (MICs)
were determined by using the twofold serial dilution tech-
nique (Rajbhandari and Schopke, 1999). The twofold serial
dilutions of the tested compound solutions were prepared.
The final concentrations of the solutions were 269.2–0.003,
295.6–0.004, and 974.6–0.013 lM for compounds 1–3,
Statistical analysis (Woodson, 1987)
All data were expressed as mean SE. Student’s t test was
applied for detecting the significance of difference between
each sample; P \ 0.05 was taken as the level of
significance.
Chemistry
Extraction and isolation
The dried powdered fruits (3.0 kg) of G. aquatica were
subjected to exhaustive extraction with 95 % EtOH (8 L
94) to yield 230 g of a solid extract, which was then
suspended in water and successively partitioned with pet-
roleum ether, EtOAc, and n-BuOH to obtain petroleum
ether (16.5 g), EtOAc (7 g), and n-BuOH (47 g) fractions
after removing solvent under reduced pressure. The
n-BuOH-soluble fraction was applied to a column of
polyamide and eluted with H2O and 25, 50, 75, and 100 %
123

Med Chem Res
MeOH. The H2O fraction (28 g) was chromatographed
over Si gel and Si gel flash CC eluted with CHCl3–MeOH
(90:10–70:30) to give four fractions of A (950 mg), B
(2.6 g), C (3.3 g), and D (4.3 g). Fraction D (4.3 g) was
chromatographed over a reversed-phase Sepralyte RP-18
CC using a gradient of MeOH–H2O (50:50–60:40) to yield
D1 (1.9 g), and D2 (2 g). Fraction D1 was rechro-
matographed over Si gel and Si gel flash CC eluted with
CHCl3–MeOH (80:20–75:25) to give four fractions of D1a
(360 mg), D1b (350 mg), D1c (210 mg), and D1d (90 mg).
Fraction D1c was purified on a reversed-phase Sepralyte
RP-18 CC using MeOH–H2O (57:43) and then finally pu-
rified by Sephadex LH-20 CC eluted with MeOH to afford
1 (50 mg). By the same method, fraction D2 was rechro-
matographed over Si gel and Si gel flash CC eluted with
CHCl3–MeOH (80:20–75:25) to give three fractions of
D2a (200 mg), D2b (425 mg), and D2c (390 mg). Fraction
D2b was purified on a reversed-phase Sepralyte RP-18 CC
using MeOH-H2O (59:41) and then finally purified by
Sephadex LH-20 CC eluted with MeOH to afford 2
(40 mg). Fraction C was rechromatographed over Si gel
CC eluted with CHCl3–MeOH (85:15–80:20) to yield C1
(800 mg), C2 (750 mg), C3 (530 mg), and C4 (420 mg).
Fraction C2 was repeatedly subjected to Si gel CC eluted
with CHCl3–MeOH (85:15) and a reversed-phase Sepralyte
RP-18 CC eluted with MeOH–H2O (80:20) and then finally
purified by Sephadex LH-20 CC eluted with MeOH to
afford 3 (45 mg).
119.99 (CH, C-12), 109.14 (CH, C-100), 102.25 (C, C-5),
86.25 (CH, C-10), 78.58 (C, C-300), 77.65 (CH, C-50), 76.25
(CH, C-30), 75.90 (CH, C-200), 73.23 (CH2, C-400), 72.25
(CH, C-20), 68.01 (CH, C-40), 65.36 (CH2, C-60), 62.40
(CH2, C-500), 39.89 (CH2, C-11), 25.29 (CH3, C-15), 17.89
(CH3, C-14).
Acid hydrolysis of compounds 1 and 2
A 5 mg of each compound was refluxed separately with
2 M HCl in MeOH (5 mL) at 80 °C for 6 h in a water bath.
The reaction mixture was evaporated, and the hydrolysate
after dilution with H2O (10 mL) was extracted with CHCl3
(3 9 10 mL). The CHCl3 extracts were evaporated to af-
ford the aglycons, which were identified as echinocystic
acid (m/z 472 by EIMS and its NMR data) from 1 and 2.
The aqueous layer was neutralized with 2 N KOH solution
and concentrated to 1 mL under reduced pressure. The
concentrated aqueous layer showed spots at the same Rf as
glucose, xylose, arabinose and rhamnose for 1 and 2 on
TLC silica gel (30:12:4 CHCl3:MeOH:H2O), 9 mL of
lower layer and 1 mL of HOAc, and on PC (iso-PrOH:n-
BuOH:H2O 7:1:2), using aniline hydrogen phthalate as a
detecting reagent.
Echinocystic acid: An amorphous solid from MeOH;
1
EIMS m/z 472 [M]?; H NMR (DMSO-d6, 500 MHz) d
5.20 (1H, brs, H-12), 4.30 (1H, brt, H-16), 3.00 (1H, dd,
J = 11.0, 5.0 Hz, H-3), 1.30 (s, H3-27), 0.90 (s, H3-30),
0.89 (s, H3-23), 0.85 (s, H3-25), 0.83 (s, H3-29), 0.68 (s,
H3-26), 0.67 (s, H3-24); 13C NMR data (DMSO-d6,
125 MHz) d 177.0 (C-28), 144.1 (C-13), 121.2 (C-12), 76.8
(C-3), 72.9 (C-16), 54.8 (C-5), 47.3 (C-17), 46.3 (C-19),
46.2 (C-9), 41.0 (C-14), 40.0 (C-18), 38.3 (C-4), 39.1
(C-8), 38.1 (C-1), 36.5 (C-10), 34.7 (C-15), 35.1 (C-21),
32.8 (C-29), 32.6 (C-7), 31.3 (C-22), 30.2 (C-20), 28.2
(C-23), 26.9 (C-2), 26.4 (C-27), 24.1 (C-30), 22.7 (C-11),
18.0 (C-6), 16.8 (C-26), 16.0 (C-24), 15.1 (C-25).
Aquaticasaponin A (1): An amorphous solid from
MeOH; IR (KBr) tmax 3445, 1740, 1690 cm-1; HRESIMS
m/z 1856.8787 [M–H]- (calcd. for C91H142NO38,
1
1856.9210); H NMR and 13C NMR data see Tables 1, 2,
and 3.
Aquaticasaponin B (2): An amorphous solid from MeOH;
IR (KBr) tmax 3450, 1735, 1690 cm-1; HRESIMS m/z
1690.7631 [M-H]- (calcd. for C81H128NO36, 1690.8216);
1H NMR and 13C NMR data see Tables 1, 2, and 3.
Aquaticine C (3): White amorphous powder from
MeOH; UV (MeOH) kmax: 220, 270 nm; IR (KBr) tmax
3440, 1635 cm-1; ESIMS m/z 514 [M ? H]?, 382
[M ? H-apiose]?, and 763 [2M ? H-2 apiose]?; 1H NMR
(DMSO-d6, 600 MHz) d 8.06 (1H, s, H-8), 5.40 (1H, d,
J = 8.5 Hz, H-10), 5.26 (1H, t, J = 6.8 Hz, H-12), 4.86
(1H, d, J = 3.1 Hz, H-100), 4.53 (2H, d, J = 6.8 Hz, H-11),
3.87 (1H, d, J = 9.3 Hz, H-400a), 3.85 (1H, d, J = 3.1 Hz,
H-200), 3.84 (1H, dd, J = 11.5, 5.5 Hz, H-60a), 3.68 (1H, m,
H-50), 3.64 (1H, dd, J = 11.5, 2.8 Hz, H-60b), 3.59 (1H, d,
J = 9.3 Hz, H-400b), 3.49 (1H, t, J = 9.3 Hz, H-40), 3.40
(1H, d, J = 11.2 Hz, H-500a), 3.34–3.32 (3H, m, H-20, H-30,
H-500b), 1.77 (3H, s, H-14), 1.64 (3H, s, H-15), 7.02, 5.55,
5.38, 5.32, 5.14, 5.11 and 4.50 (brs, NH and OHs); 13C
MNR (DMSO-d6, 100 MHz) d 154.67 (C, C-2), 153.30 (C,
C-4), 153.16 (C, C-6), 142.91 (CH, C-8), 134.59 (C, C-13),
Acid hydrolysis of compound 3
5 milligram of 3 was hydrolyzed with 0.1 N H2SO4
(1.0 mL) at 100 8C for 2 h. After neutralization with
NaHCO3, solvent was evaporated. The water-soluble part
of the residue showed a spot at the same Rf as glucose and
apiose for 3 on TLC (silica gel 14:6:1 CHCl3–MeOH–
H2O).
Results and discussion
Dried fruits of G. aquatica were extracted with ethanol,
and then the dried ethanolic extract was suspended in water
and fractionated with petroleum ether, ethyl acetate, and
123

Med Chem Res
1
Table 1 1H and 13C NMR data (d in ppm) for the aglycon moieties of compounds 1 and 2 (500 MHz for H and 125.0 MHz for 13C)
Position
1
2
dCa
dHa
dHb
dCa
dHa
dHb
1
38.14
1.46, 0.74
1.79, 1.55
2.99
1.63, 0.95
1.90, 1.65
3.10
38.15
1.48, 0.76
1.80, 1.55
2.99
1.64, 0.98
2
25.54
88.15
38.18
55.12
18.07
32.41
38.45
46.09
36.28
22.95
121.46
143.41
41.91
34.82
72.51
48.19
39.72
46.65
30.15
34.95
30.73
27.55
16.31
15.36
16.60
26.18
174.85
32.91
24.21
25.50
88.14
38.22
55.08
17.96
32.43
38.45
46.09
36.28
22.95
121.44
143.38
41.91
34.85
72.51
48.17
39.74
46.64
30.16
34.96
30.68
27.55
16.32
15.35
16.60
26.19
174.85
32.87
24.21
1.90, 1.65
3
3.10
4
–
–
–
–
5
0.64
0.75
0.62
0.76
6
1.39, 1.21
1.33, 1.10
–
1.55, 1.30
1.45, 1.20
–
1.41, 1.23
1.34, 1.12
–
1.55, 1.31
7
1.46, 1.20
8
–
9
1.45
1.60
1.46
1.61
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
–
–
–
–
1.76
1.88
1.76
1.89
5.16, brs
–
5.28, brs
–
5.14, brs
–
5.29, brs
–
–
–
–
–
1.56, 1.20
4.32
1.61, 1.45
4.47
1.56, 1.21
4.30
1.62, 1.45
4.47
–
–
–
2.80
2.90
2.81
2.92
2.20, 0.94
–
2.30, 1.05
–
2.21, 0.95
–
2.30, 1.05
–
1.82, 1.05
1.84, 1.55
0.85, s
0.61, s
0.83, s
0.61, s
1.27, s
–
1.90, 1.17
1.95, 1.72
0.96, s
0.75, s
0.92, s
0.74, s
1.34, s
–
1.83, 1.07
1.86, 1.56
0.85, s
0.60, s
0.83, s
0.62, s
1.28, s
–
1.91, 1.19
1.93, 1.71
0.96, s
0.76, s
0.93, s
0.75, s
1.35, s
–
0.80, s
0.83, s
0.85, s
0.93, s
0.81, s
0.84, s
0.86, s
0.94, s
The assignment was based upon DEPT, DQF-COSY, HMQC, and HMBC experiments
a
DMSO-d6
b
CD3OD
n-butanol. The n-butanol fraction was subjected to subse-
quent purification using several chromatographic tech-
niques (polyamide, repeated silica gel, RP-18, and
Sephadex LH-20 columns); two new and one known
compounds were isolated (Fig. 1).
echinocystic acid. The 13C-NMR spectrum of 1 showed 91
carbon signals (Tables 1, 2, 3), from which 30 signals were
attributed to the aglycon and 41 signals were attributed to the
sugar moieties. The remaining 20 signals were consistent
with the presence of two monoterpene carboxylic acids. It
was apparent from the chemical shifts of the C-3 (d 88.15)
and C-28 (d 174.85) that 1 was a bisdesmosidic glycoside. In
Aquaticasaponin A (1) was obtained as a white amor-
phous solid. The molecular formula was deduced as
C91H143NO38 from an [M-H]- ion at m/z 1856.8787 in the
1
the H-NMR spectrum of 1, seven sugar anomeric proton
1
HRESIMS and from the 13C-NMR spectrum. The H and
signals appeared at d 4.21 (1H, d, J = 5.7 Hz, ara), 4.24 (1H,
d, J = 8.0 Hz, glc-NAc), 4.34 (1H, d, J = 7.4 Hz, xyl0),
4.43 (1H, d, J = 7.4 Hz, xyl), 4.69 (1H, br s, rha0), 5.11 (1H,
br s, rha), and 5.29 (1H, d, J = 7.2 Hz, glc). The corre-
sponding seven anomeric carbons were observed at d 103.23,
13C-NMR spectra displayed resonances due to the seven
tertiarymethyl groups, and the two olefinic carbonsindicated
the aglycon of 1 possessed an olean-12-ene skeleton. After
an extensive 2D NMR study, the aglycon was identified as
123

Med Chem Res
1
Table 2 1H and 13C-NMR data (d in ppm) for the sugar moieties of compounds 1 and 2 (500 MHz for H and 125.0 MHz for 13C)
Position
1
2
dCa
dHa
dHb
dCa
dHa
dHb
C3-Glc-Nac
1
2
103.54
55.70
73.71
70.74
73.71
68.10
–
4.24, d, 8.0
3.41
4.44, d, 8.5
3.66, m
3.44
103.53
55.69
73.72
70.74
73.72
68.09
–
4.25, d, 8.0
3.40
4.43, d, 8.2
3.65
3
3.26
3.28
3.44
4
3.03
3.32
3.05
3.32
5
3.22
3.42
3.23
3.42
6
3.48, 3.88
7.72, d, 9.2
–
3.71, 4.07
–
3.49, 3.91
7.72, d, 9.2
–
3.71, 4.07
–
NH
CO
CH3
Ara
1
168.92
23.18
–
168.84
23.19
–
1.76, s
1.96, s
1.77, s
1.96, s
103.23
70.55
72.28
68.02
64.63
4.21, d, 5.7
3.31
4.33, d, 6.6
3.58
103.23
70.55
72.25
68.01
64.62
4.22, d, 5.7
3.32
4.33, d, 6.3
3.58
2
3
3.32
3.51
3.34
3.51
4
3.58
3.78
3.60
3.78
5
3.30, 3.67
3.52, 3.84
3.31, 3.66
3.52, 3.84
C28-Glc
1
2
3
4
5
6
92.78
75.46
77.18
69.52
76.01
65.79
5.29, d, 7.2
3.34
5.35, d, 7.2
3.58
92.82
75.47
77.14
69.52
76.03
65.79
5.30, d, 7.2
3.34
5.35, d, 7.0
3.58
3.22
3.46
3.24
3.46
3.40
3.61
3.41
3.61
3.24
3.51
3.25
3.51
3.71, 3.14
3.92, 3.26
3.71, 3.16
3.92, 3.26
Rha
1
99.99
69.64
70.55
83.01
67.18
17.95
5.11, brs
3.74
5.39, brs
3.95
100.01
69.68
70.55
82.98
67.17
17.95
5.12, brs
3.76
5.39, brs
3.95
2
3
3.64
3.85
3.62
3.85
4
3.38
3.53
3.37
3.52
5
3.52
3.80
3.53
3.80
6
1.17, d, 6.3
1.32, d, 6.3
1.17, d, 6.0
1.32, d, 5.8
Xyl
1
104.92
73.49
86.33
69.30
65.43
4.43, d, 7.4
3.22
4.53, d, 7.5
3.48
104.96
73.49
86.24
69.38
65.46
4.44, d, 7.2
3.23
4.53, d, 7.5
3.48
2
3
3.36
3.47
3.38
3.47
4
3.26
3.50
3.27
3.50
5
3.70, 3.02
3.22, 3.89
3.72, 3.03
3.22, 3.89
Xyl0
1
104.60
73.83
76.01
69.30
65.79
4.34, d, 7.4
3.07
4.46, d, 7.2
3.35
104.59
73.83
76.10
69.30
65.79
4.34, d, 7.4
3.07
4.46, d, 7.2
3.34
2
3
3.12
3.52
3.13
3.52
4
3.25
3.48
3.27
3.48
5
3.71, 3.10
3.90, 3.33
3.73, 3.10
3.90, 3.33
Rha0
1
96.57
69.73
71.50
69.42
4.69, brs
5.05
4.85, d, 1.5
5.27, dd, 3.5, 1.5
5.13, dd, 9.8, 3.5
3.54
96.50
72.10
69.21
72.28
4.52, brs
4.93
4.71, brs
5.06
2
3
4.94
3.62
3.87
4
3.36
3.18
3.40
123

Med Chem Res
Table 2 continued
Position
1
2
dCa
dHa
dHb
dCa
dHa
dHb
5
6
68.25
17.95
3.61
3.78
68.22
17.91
3.46
3.63
1.18, d, 6.0
1.29, d, 6.3
1.16, d, 6.0
1.25, d, 6.0
The assignment was based upon DEPT, DQF-COSY, HMQC, and HMBC experiments
a
DMSO-d6
b
CD3OD
1
Table 3 1H and 13C NMR data (d in ppm) for the monoterpenic acid moieties of compounds 1 and 2 (500 MHz for H and 125 MHz for 13C)
Position
1
2
dCa
dHa
dHb
dCa
dHa
dHb
MT1
1
2
3
4
5
6
7
8
166.31
126.61
142.95
23.11
–
–
166.06
126.56
142.77
23.09
–
–
–
–
–
–
6.52, t, 6.9
2.17–2.05, m
1.47–1.35, m
–
6.83, t, 7.6
2.32–2.20, m
1.62–1.52, m
–
6.53, t, 7.6
2.18–2.09, m
1.48–1.42, m
–
6.80, t, 7.6
2.30–2.25, m
1.60–1.55, m
–
40.59
40.58
71.33
71.32
145.66
111.39
5.88, dd, 17.2, 10.5
4.98, dd, 10.6, 1.9
5.16, dd, 17.2, 1.9
1.65, s
5.89, dd, 17.3, 10.7
5.07, dd, 11.0, 1.2
5.23, dd, 17.3, 1.6
1.75, s
145.68
111.38
5.85, dd, 17.2, 10.6
4.98, dd, 10.6, 1.9
5.17, dd, 17.2, 1.9
1.74, s
5.90, dd, 17.3, 10.7
5.05, dd, 10.7, 2.2
5.22, dd, 17.3, 2.2
1.85, s
9
10
MT2
1
12.18
27.80
12.18
27.81
1.14, s
1.28, s
1.14, s
1.27, s
166.99
126.44
143.61
23.26
–
–
2
–
–
3
6.69, t, 7.9
2.17–2.05, m
1.47–1.35, m
–
6.67, t, 7.6
2.32–2.20, m
1.62–1.52, m
–
4
5
40.59
6
71.33
7
145.66
111.39
5.82, dd, 17.2, 10.5
4.96, dd, 10.6, 1.9
5.14, dd, 17.2, 1.9
1.73, s
5.87, dd, 17.3, 10.7
5.04, dd, 11.0, 1.2
5.20, dd, 17.3, 1.6
1.83, s
8
9
12.00
27.80
10
1.15, s
1.25, s
The assignment was based upon DEPT, DQF-COSY, HMQC, and HMBC experiments
a
DMSO-d6
b
CD3OD
103.54, 104.60, 104.92, 96.57, 99.99, and 92.78, respectively
(Table 2). The presence of two methyl signals at dC 17.95, dH
1.17 (d, J = 6.3 Hz) and dC 17.95, dH 1.18 (d, J = 6.0 Hz)
L-arabinose, andL-rhamnosewhichwere identifiedbyco-TLC
and PC analysis with authentic sugars. The structures of the
oligosaccharide moieties were determined through DQF-
COSY, HMQC, and HMBC experiments. Starting from the
well-resolved anomeric proton signals or the methyl group
proton signals for the deoxy sugars or the acylated proton
signals of the acylated sugar, the sequential assignments of
all the proton resonances to individual monosaccharide were
1
in the H and 13C-NMR spectra suggested that two of the
seven monosaccharides are being deoxy sugar. Acid hy-
drolysisof 1 afforded echinocystic acid, which was identified
by comparison with the reference data (Akai et al., 1985,
Maillard et al., 1992) and with the D-glucose, D-xylose,
123

Med Chem Res
Table 4 Antimicrobial activity as MICs (lM) of compounds 1–3
Tested microorganisms
MIC (lM)
1
2
3
Standard
G ?ve bacteria
S. pneumonia
B. subtilis
Ampicillin
36.9
4.6
67.3
8.4
30.5
15.2
1.4
0.17
G -ve bacteria
P. aeruginosa
E. coli
Gentamycin
NA
NA
NA
13.5
73.9
67.3
121.8
27.1
Fungi
Amphotericin B
A. fumigates
S. racemosum
G. candidum
C. albicans
73.9
9.2
67.3
67.3
33.7
NA
60.9
60.9
30.5
NA
1.1
16.9
0.12
0.25
18.5
NA
NA no activity
Table 5 Cytotoxicity of compounds 1–3 against cultured MCF-7 and
HCT-116 cancer cell lines
achieved using DQF-COSY spectrum. On the basis of the
assigned proton signals, a HMQC experiment then gave the
corresponding carbon assignments, and these were further
clarified by HMBC experiment. Accordingly, the assign-
ments of the protons and protonated carbons were estab-
lished (Table 2), and the seven sugar units were identified as
one glucose, two xyloses, two rhamnoses, one arabinose, and
one N-acetylglucosamine. The 13C-NMR data for the sugar
moieties indicated that all the monosaccharides were in the
pyranose forms. The anomeric configurations for the sugar
moieties were fully defined from their chemical shifts and
3JH1,H2 coupling constants (Table 2). Accordingly, the glu-
copyranosyl unit, the two xylopyranosyl units, and the
N-acetylglucosamine unit were established to be in the
b-configuration, while the arabinopyranosyl unit and the two
rhamnopyranosyl units were in the a-configuration (Gorin
and Mazurek, 1975; Overend, 1972). The linkage of the
sugar units at C-3 was established from the following HMBC
correlation: H-1 (d 4.21) of Ara with C-6 (d 68.10) of Glc-
NAc. The attachment of the disaccharide moiety to C-3 of the
aglycon was confirmed by the long-range correlation be-
tween H-1 (d 4.24) of Glc-NAc and C-3 (d 88.15) of the
aglycon. The sequence of the sugar chin at C-28 was deduced
from the following HMBC correlations: H-1(d 5.11) of Rha
with C-2 (d 75.46) of Glc; H-1 (d 4.43) of xyl with C-4 (d
83.01) of Rha; H-1 (d 4.34) of xyl0 with C-3 (d 86.33) of xyl;
and H-1 (d 4.69) of Rha0 with C-6 (d 65.79) of Glc, while the
attachment of the pentasaccharide chain to C-28 of the
aglycon was based on a correlation of H-1 (d 5.29) of Glc
with the C-28 (d 174.85) of the aglycon. The presence of two
monoterpenic units in 1 was indicated by various NMR data
and by comparison of these data with those reported in the
literature (Zang et al., 1999a, b, c, Okada et al., 1980), which
confirmed that both monoterpenoid moieties in 1 were the
same and were characterized as (2E)-6-hydroxy-2,6-
Compounds
Growth inhibition constant (IC50)a (lM)
MCF-7
HCT-116
1
0.80 0.3
1.0 0.3
[10
0.5 0.21
0.76 0.4
[10
2
3
Doxorubicinb
0.81 0.06
0.86 0.1
a
IC50 is defined as the concentration that resulted in a 50 % decrease
in cell number, and the results are mean standard deviation of three
independent replicates. The IC50 [ 10 lM was considered to be no
cytotoxicity
b
Positive control substance
dimethyl-2,7-octadienoic acid. The stereochemistry of the
D2,3 double bond was determined as E from the chemical
shifts of H-3, since the olefinic proton of the Z-isomer ap-
peared at a higher field (Okada et al., 1980). The absolute
configuration of C-6 position was not established. The
binding sites of the two monoterpenic acids MT1 and MT2
were revealed by two acylation shifts observed at d 5.05 and
4.92. Using both COSY and HMBC experiments, these
signals were assigned to H-2 and H-3 of Rha0, respectively.
Further, the HMBC spectrum exhibited significant cross-
correlations between H-2 of Rha0 and the carbonyl carbon
(d 166.31) of the monoterpenic acid MT1 and between H-3 of
Rha0 and the carbonyl carbon (d 166.99) of the monoterpenic
acid MT2. Therefore, the two monoterpenic acids MT1 and
MT2 were located at C-2 and C-3 of Rha0, respectively.
The foregoing evidence led to the elucidation of the
structure of compound 1 as 3-O-a-L-arabinopyranosyl-
(1 ? 6)-2-acetamido-2-deoxy-b-D-glucopyranosyl echinocys-
tic acid 28-O-b-D-xylopyranosyl-(1 ? 3)-b-D-xylopyranosyl-
123

Med Chem Res
(1 ? 4)-a-L-rhamnopyranosyl-(1 ? 2)-[(2E)-6-hydroxy-2,6-
dimethyl-2,7-octadienoyl-(1 ? 2) and (20E)-60-hydroxy-
20,60-dimethyl-20,70-octadienoyl-(1 ? 3)-a-L-rhamnopyra-
nosyl-(1 ? 6)]-b-D-glucopyra-nosyl ester.
activity against S. racemosum with an MIC 9.2 lM (nearly
twofold lower than that of amphotericin B), followed by
compound 2 against the G -ve bacteria E. coli with an
MIC 67.3 lM (nearly only twofold greater than that of
gentamycin). Compound 3 showed the lowest level of
activity against all strains. Compounds 1 and 2 exhibited
good cytotoxicity against breast cancer (MCF-7) and colon
cancer (HCT-116) cell lines and gave IC50 in the range
0.5–1.0 lM. On the other hand, compound 3 showed no
cytotoxicity against these cancer cell lines ([10 lM)
(Table 5).
Aquaticasaponin B (2), a white amorphous solid, gave
an [M-H]- ion at 1690.7631 in the HRESIMS, consistent
with a molecular formula of C81H129NO36, 166 mass units
lower than that of 1, implying 2 was a derivative of 1 with
one less monoterpenic acid. Acid hydrolysis of 2 allowed
the identification of the same aglycon (echinocystic acid)
and the same sugar components as 1, that is, D-glucose,
D-xylose, L-arabinose, and L-rhamnose based on the TLC
and PC analysis. Detailed analysis of the 13C-NMR data
obtained for 2 indicated the chemical shifts for the aglycon
part and sugar moieties of 2 bore a close resemblance to
those of 1, suggesting that both compounds had the same
aglycon and the same sugar structures and sequence at both
the C-3 and C-28 positions (Tables 1, 2). A comparison of
the NMR spectroscopic data for the acylated oligosaccha-
ride chain at C-28 of 1 and 2 showed that 2 lacked the
monoterpenic acid MT2 connected to C-3 of Rha0 as found
in 1 and the remaining monoterpenic acid MT1 connected
to C-2 of Rha0 was identified as (2E)-6-hydroxy-2,6-
dimethyl-2,7-octadienoic acid. As observed in the HMBC
spectrum, the long-range correlation of H-2 (d 4.93) of
Rha0 with C-1 (d 166.06) of the monoterpenic acid MT1
established that the monoterpenic acid MT1 was attached to
C-2 of Rha0. The downfield shift of H-2 of Rha0 also
indicated it was the position of acylation (Table 3). On the
basis of above evidence, the structure of compound 2 was
concluded to be 3-O-a-L-arabinopyranosyl-(1 ? 6)-2-ac-
etamido-2-deoxy-b-D-glucopyranosyl echinocystic acid 28-
O-b-D-xylopyranosyl-(1 ? 3)-b-D-xylopyranosyl-(1 ? 4)-
a-L-rhamnopyranosyl-(1 ? 2)-[(2E)-6-hydroxy-2,6-dime-
thyl-2,7-octadienoyl-(1 ? 2)-a-L-rhamnopyranosyl-(1 ? 6)]-
b-D-glucopyranosyl ester.
Acknowledgments Many thanks are due to Dr. Mohammed Hosny,
Professor of Pharmacognosy, Faculty of Pharmacy, Al-Azhar
University, Cairo, Egypt and Dr. Ehab M. Mostafa, Lecturer of
Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Cairo,
Egypt, for 2D NMR and Ms Facilities.
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123