Steroidal saponins from the leaves of Cordyline fruticosa (L.) A. Chev. and their cytotoxic and antimicrobial activity

Abstract

Three new steroidal saponins, spirosta-5,25(27)-diene-1β,3β-diol-1-O-α-l-rhamnopyranosyl-(1→2)-β-d-fucopyranoside (fruticoside H) 1, 5α-spirost-25(27)-ene-1β,3β-diol-1-O-α-l-rhamnopyranosyl-(1→2)-(4-O-sulfo)-β-d-fucopyranoside (fruticoside I) 2, and (22S)-cholest-5-ene-1β,3β,16β,22-tetrol 1-O-β-galactopyranosyl-16-O-α-l-rhamnopyranoside (fruticoside J) 3, together with the known quercetin 3-O-β-d-glucopyranoside, quercetin 3-O-[6-trans-p-coumaroyl]-β-d-glucopyranoside, quercetin 3-rutinoside, apigenin 8-C-β-d-glucopyranoside and farrerol, were isolated from the leaves of Cordyline fruticosa. Their structures were elucidated by spectroscopic techniques (1H NMR, 13C NMR, HSQC, 1H–1H COSY, HMBC, TOCSY, NOESY), mass spectrometry (HRESIMS, Tandem MS–MS), chemical methods and by comparison with published data. Compounds 1 and 2 showed moderate cytotoxic activity against MDA-MB 231 human breast adenocarcinoma cell line, HCT 116 human colon carcinoma cell line, and A375 human malignant melanoma cell line, while compound 3 was not active. Compound 2 also showed a moderate antibacterial activity against the Gram-positive Enterococcus faecalis

Full text

Steroidal
saponins
from
the
leaves
of
Cordyline
fruticosa
(L.)
A.
Chev.
and
their
cytotoxic
and
antimicrobial
activity
Romuald
T.
Fouedjou
a
,
Re
´my
B.
Teponno
a
,
Luana
Quassinti
c
,
Massimo
Bramucci
c
,
Dezemona
Petrelli
d
,
Luca
A.
Vitali
c
,
Dennis
Fiorini
b
,
Le
´on
A.
Tapondjou
a,
*,
Luciano
Barboni
b,
**
a
Department
of
Chemistry,
Faculty
of
Science,
University
of
Dschang,
Box
183,
Dschang,
Cameroon
b
School
of
Science
and
Technology,
Chemistry
Division,
University
of
Camerino,
Via
S.
Agostino
1,
I-62032
Camerino,
Italy
c
School
of
Pharmacy,
University
of
Camerino,
Via
Gentile
III
da
Varano,
I-62032
Camerino,
Italy
d
School
of
Biosciences
and
Biotechnology,
University
of
Camerino,
Via
Gentile
III
da
Varano,
I-62032
Camerino,
Italy
1.
Introduction
Cordyline
fruticosa
(L.)
A.
Chev.
(syn.
C.
terminalis
(L.)
Kunth)
belongs
to
the
family
Agavaceae
(IPNI,
2012),
which
comprises
more
than
480
species
distributed
in
tropical
and
subtropical
regions
of
the
World
(Yokosuka
et
al.,
2000).
The
plant
is
widely
used
in
both
everyday
life
and
traditional
medicine.
A
hedge
of
C.
fruticosa
around
the
house
was
believed
to
ward
off
evil
spirits
and
bring
good
luck
(Elbert
and
Roger,
1989).
The
leaves
have
been
used
for
costumes,
decorations,
clothing,
sandals,
packaging,
and
cooking.
The
rhizomes
can
be
baked
into
a
molasses-like
food
product
and
eaten
(Hinkle,
2007).
In
addition,
the
plant
is
traditionally
used
for
the
treatment
of
various
diseases:
the
leaves
are
used
to
treat
sore
throat
and
neck
pain
(Whistler,
1985),
as
haemostatic
(Buttner,
2001;
Dalimartha,
2007),
and
to
induce
abortion
(Nugent,
2006);
the
roots
are
used
for
toothache,
laryngitis
and
infections
of
mammary
glands
(Dalimartha,
2007;
Nombo
and
Leach,
2010).
The
plants
of
the
genus
Cordyline
are
very
well
known
as
source
of
steroidal
saponins
and
cholestane
glycosides
(Simmons-Boyce
and
Tinto,
2007;
Challinor
and
De
Voss,
2013;
Yang
et
al.,
1989;
Mimaki
et
al.,
1997,
1998).
Saponins
possessing
a
steroidal
(C27)
skeleton
are
commonly
found
in
monocotyledonous
angiosperms,
especially
plants
of
the
yam
(Dioscoreaceae),
asparagus
(Aspar-
agaceae),
solanum
(Solanaceae),
lily
(Liliaceae),
onion
(Amarylli-
daceae),
and
agave
(Agavaceae)
families
(Challinor
and
De
Voss,
2013;
Simmons-Boyce
and
Tinto,
2007).
Steroidal
saponins
can
be
divided
into
three
structural
subclasses,
spirostane-,
furostane-
and
cholestane-type
(open
chain)
glycosides
(Challinor
and
De
Voss,
2013)
and,
over
the
years,
have
attracted
attention
for
the
wide
spectrum
of
their
biological
actions,
particularly
cytotoxic,
antimicrobial,
antifungal
and
in
vivo
antitumoral
activities
(Sautour
et
al.,
2007;
Li
et
al.,
2012).
Cytotoxic
activity
of
spirostane
and
open-chain
saponins
isolated
from
C.
fruticosa
has
been
recently
reported
(Yokosuka
et
al.,
2012;
Bogoriani
et
al.,
2007).
In
our
continuous
search
of
potentially
interesting
bioactive
saponins
from
Cameroonian
medicinal
plants
(Nzowa
et
al.,
2010;
Phytochemistry
Letters
7
(2014)
62–68
A
R
T
I
C
L
E
I
N
F
O
Article
history:
Received
29
July
2013
Received
in
revised
form
28
September
2013
Accepted
3
October
2013
Available
online
22
October
2013
Keywords:
Cordyline
fruticosa
Agavaceae
Steroidal
saponins
Biological
activity
A
B
S
T
R
A
C
T
Three
new
steroidal
saponins,
spirosta-5,25(27)-diene-1
b
,3
b
-diol-1-O-
a
-
L
-rhamnopyranosyl-(1!2)-
b
-
D
-fucopyranoside
(fruticoside
H)
1,
5
a
-spirost-25(27)-ene-1
b
,3
b
-diol-1-O-
a
-
L
-rhamnopyranosyl-
(1!2)-(4-O-sulfo)-
b
-
D
-fucopyranoside
(fruticoside
I)
2,
and
(22S)-cholest-5-ene-1
b
,3
b
,16
b
,22-tetrol
1-O-
b
-galactopyranosyl-16-O-
a
-
L
-rhamnopyranoside
(fruticoside
J)
3,
together
with
the
known
quercetin
3-O-
b
-
D
-glucopyranoside,
quercetin
3-O-[6-trans-p-coumaroyl]-
b
-
D
-glucopyranoside,
quer-
cetin
3-rutinoside,
apigenin
8-C-
b
-
D
-glucopyranoside
and
farrerol,
were
isolated
from
the
leaves
of
Cordyline
fruticosa.
Their
structures
were
elucidated
by
spectroscopic
techniques
(
1
H
NMR,
13
C
NMR,
HSQC,
1
H–
1
H
COSY,
HMBC,
TOCSY,
NOESY),
mass
spectrometry
(HRESIMS,
Tandem
MS–MS),
chemical
methods
and
by
comparison
with
published
data.
Compounds
1
and
2
showed
moderate
cytotoxic
activity
against
MDA-MB
231
human
breast
adenocarcinoma
cell
line,
HCT
116
human
colon
carcinoma
cell
line,
and
A375
human
malignant
melanoma
cell
line,
while
compound
3
was
not
active.
Compound
2
also
showed
a
moderate
antibacterial
activity
against
the
Gram-positive
Enterococcus
faecalis.
ß
2013
Phytochemical
Society
of
Europe.
Published
by
Elsevier
B.V.
All
rights
reserved.
*Corresponding
author.
Tel.:
+237
75004826;
fax:
+237
33451735.
** Corresponding
author.
Tel.:
+39
0737402240;
fax:
+39
0737637345.
E-mail
addresses:
tapondjou2001@yahoo.fr
(L.A.
Tapondjou),
luciano.barboni@unicam.it
(L.
Barboni).
Contents
lists
available
at
ScienceDirect
Phytochemistry
Letters
jo
u
rn
al
h
om
ep
ag
e:
ww
w.els
evier.c
o
m/lo
c
ate/p
hyt
ol
1874-3900/$
–
see
front
matter
ß
2013
Phytochemical
Society
of
Europe.
Published
by
Elsevier
B.V.
All
rights
reserved.
http://dx.doi.org/10.1016/j.phytol.2013.10.001

Ponou
et
al.,
2010;
Tapondjou
et
al.,
2011),
we
have
examined
the
methanol
extract
from
the
leaves
of
C.
fruticosa
growing
in
the
western
highlands
of
Cameroon.
In
the
present
paper
we
report
the
isolation
and
structure
elucidation
of
three
new
steroidal
saponins:
fruticosides
H
(1)
and
I
(2),
having
a
spirostane-type
aglycone,
and
the
open
chain
(alliosterol-type)
fruticoside
J
(3)
(Fig.
1).
The
known
quercetin
3-O-
b
-
D
-glucopyranoside,
quercetin
3-O-[6-
trans-p-coumaroyl]-
b
-
D
-glucopyranoside,
quercetin
3-rutinoside,
apigenin
8-C-
b
-
D
-glucopyranoside
(vitexin)
and
farrerol
were
also
isolated.
Since,
as
stated
above,
steroidal
saponins
are
known
to
possess
cytotoxic
and
antimicrobial
properties,
compounds
1–3
were
investigated
for
their
cytotoxic
activity
against
three
tumour
cell
lines
namely
the
MDA-MB
231
human
breast
adenocarcinoma
cell
line,
HCT
116
human
colon
carcinoma
cell
line,
and
A375
human
malignant
melanoma
cell
line,
and
for
their
antimicrobial
activity
against
Staphylococcus
aureus,
Escherichia
coli,
Pseudomonas
aeruginosa,
Enterococcus
faecalis,
and
Candida
albicans.
2.
Results
and
discussion
2.1.
Structure
elucidation
Column
chromatography
of
the
EtOAc
soluble
fraction
of
the
MeOH
extract
from
the
dried
leaves
of
C.
fruticosa
led
to
the
isolation
and
structure
elucidation
of
three
new
steroidal
saponins,
to
which
we
gave
the
trivial
names
fruticosides
H
(1),
I
(2)
and
J
(3).
The
known
quercetin
3-O-
b
-
D
-glucopyranoside
(isoquercitrin)
(Teponno
et
al.,
2006),
quercetin
3-O-[6-trans-p-coumaroyl]-
b
-
D
-glucopyranoside
(helichrysoside)
(Lavault
and
Richomme,
2004),
quercetin
3-rutinoside
(Kazuma
et
al.,
2003),
apigenin
8-
C-
b
-
D
-glucopyranoside
(El-Toumy
et
al.,
2011)
and
farrerol
(Chen
et
al.,
2009)
were
also
isolated,
and
their
structures
elucidated
by
comparison
with
data
reported
in
the
literature.
Compound
1
was
isolated
as
a
white
amorphous
powder
from
EtOAc–MeOH.
Its
positive
ion
mode
HRESIMS
showed
the
ion
peak
at
m/z
743.3986
[M+Na]
+
,
corresponding
to
the
molecular
formula
C
39
H
60
O
12
Na.
This
was
confirmed
by
ESIMS,
which
displayed
an
ion
peak
at
m/z
743
[M+Na]
+
.
The
IR
(KBr)
spectrum
of
1
displayed
the
absorptions
characteristic
for
hydroxyl
groups
(3300
cm
1
)
and
a
spirostanol
moiety
(987,
918,
885,
833
cm
1
)
(Xu
et
al.,
2010).
The
1
H
NMR
spectrum
of
1
showed
signals
for
three
steroid
methyl
groups
at
d
1.08
(s,
Me-19),
0.95
(d,
7.0,
Me-21)
and
0.83
(s,
Me-18),
an
exomethylene
group
with
protons
at
d
4.76
(br
s)
and
4.73
(br
s)
(H-27),
an
olefinic
proton
at
d
5.55
(br
d,
5.6,
H-6),
as
well
as
two
anomeric
proton
signals
at
d
5.29
(br
d,
1.8,
H-1
00
)
and
4.26
(d,
7.7,
H-1
0
).
These
proton
signals
together
with
a
distinctive
quaternary
carbon
signal
at
d
110.8
(C-22),
showing
HMBC
correlations
with
methylene
protons
at
d
3.81
(br
d,
11.6,
H-
26a)
and
4.26
(br
d,
11.6,
H-26b),
and
other
carbon
signals
at
d
145.3
(C-25),
139.7
(C-5),
126.1
(C-6)
and
109.1
(C-27),
indicated
the
presence
of
a
spirosta-5,25(27)-diene
type
aglycone
(Xu
et
al.,
2010;
Kougan
et
al.,
2010;
Mimaki
et
al.,
1998,
1996).
Moreover,
in
the
aglycone
part
there
were
signals
at
d
3.32
(m,
H-1)
and
d
3.30
(m,
H-3)
giving
in
the
HSQC
spectrum
correlations
to
carbons
at
d
85.2
(C-1)
and
69.4
(C-3),
respectively
(Table
1).
Based
on
the
above
findings,
the
aglycone
moiety
of
1
was
recognized
to
be
spirosta-5,25(27)-diene-1
b
,3
b
-diol
(neorusco-
genin).
This
was
further
confirmed
by
correlations
observed
in
the
1
H–
1
H
COSY,
HSQC
and
HMBC
spectra
and
was
in
full
agreement
with
the
literature
data
(Perrone
et
al.,
2009;
Mimaki
et
al.,
1996).
The
alpha
configuration
of
H-1
and
H-3
was
also
confirmed
by
the
NOESY
correlation
between
H-1
(
d
3.32)
and
H-9
(
d
1.23)
and
between
beta
H-2
(
d
1.68)
and
H-19
(
d
1.08),
and
the
beta
H-2
splitting
and
coupling
constants
(br
t,
11.5
Hz).
The
sugar
part
of
the
1
H
NMR
spectrum
of
compound
1
showed
the
H-1
0
and
H-1
00
signals
at
d
4.26
and
5.29,
respectively,
which
gave
HSQC
correlations
with
carbons
at
d
101.0
(C-1
0
)
and
101.6
(C-1
00
).
The
ring
protons
of
each
monosaccharide
were
assigned
starting
from
the
anomeric
protons
using
1D-TOCSY
and
1
H–
1
H
COSY
spectra.
Finally,
the
two
deoxypyranosyl
methyls
were
observed
at
d
1.24
(d,
6.3,
H-6
00
)
and
1.23
(d,
6.2,
H-6
00
).
After
assignments
of
the
proton
signals,
the
13
C
NMR
resonances
of
each
sugar
unit
were
identified
by
HSQC
and
further
confirmed
by
HMBC
(Table
1).
Evaluation
of
spin–spin
couplings
and
chemical
shifts
as
well
as
the
comparison
with
literature
data
(Agrawal,
1992)
confirmed
the
identification
of
one
2-substituted
b
-fucopyrano-
syl
and
one
terminal
a
-rhamnopyranosyl
units.
The
coupling
constants
observed
for
the
anomeric
protons
suggested
that
the
anomeric
configurations
of
fucose
(J
=
7.7)
and
rhamnose
(J
=
1.8)
were
of
the
b
and
a
forms,
respectively.
The
sugar
sequence
and
its
linkage
site
to
the
aglycone
moiety
were
determined
from
the
HMBC
and
NOESY
spectra.
In
the
HMBC
spectrum,
interesting
correlations
were
observed
between
H-1
00
(
d
5.29)
and
C-2
0
(
d
75.1),
and
between
H-1
0
(
d
4.26)
and
C-1
(
d
85.2).
On
acid
hydrolysis,
compound
1
yielded
fucose
and
rhamnose,
which
were
identified
by
TLC
comparison
with
authentic
samples.
The
absolute
configuration
of
sugar
residues
was
determined
by
GC
analysis
of
their
chiral
derivatives.
Accordingly,
compound
1
was
elucidated
as
spirosta-5,25(27)-diene-1
b
,3
b
-diol-1-O-
a
-
L
-
rhamnopyranosyl-(1!2)-
b
-
D
-fucopyranoside,
a
new
naturally
occurring
steroidal
saponin
to
which
we
gave
the
trivial
name
of
fruticoside
H.
Compound
2
was
obtained
as
a
white
amorphous
powder
from
EtOAc–MeOH.
Its
negative
ion
mode
HRESIMS
showed
the
quasimolecular
ion
peak
at
m/z
801.3727
[MH]

,
corresponding
to
the
molecular
formula
C
39
H
62
O
15
S.
This
was
confirmed
by
the
negative
ion
mode
ESIMS
which
showed
the
quasimolecular
ion
peak
at
m/z
801
[MH]

.
The
1
H
NMR
spectrum
of
2
exhibited
signals
for
two
methyl
singlets
at
d
0.81
(Me-18),
0.93
(Me-19),
three
methyl
doublets
at
d
0.95
(7.0,
Me-21),
1.24
(6.0,
H-6
00
)
and
1.29
(6.3,
H-6
0
),
an
exomethylene
group
with
protons
at
d
4.77
(br
s)
and
4.73
(br
s)
(H-27),
as
well
as
two
anomeric
proton
signals
at
d
5.34
(br
d,
1.8,
H-1
00
)
and
4.36
(d,
7.6,
H-1
0
).
The
13
C
NMR
spectrum
showed
39
signals
(Table
1)
among
which
27
were
assigned
to
the
aglycone;
the
remaining
12
signals,
including
two
anomeric
Fig.
1.
Structures
of
compounds
1–3.
R.T.
Fouedjou
et
al.
/
Phytochemistry
Letters
7
(2014)
62–68
63

carbons
at
d
100.1
(C-1
0
)
and
101.6
(C-1
00
),
were
indicative
of
the
presence
of
two
hexose
moieties.
The
1
H
and
13
C
NMR
data
of
the
compound
2
were
similar
to
those
of
1,
the
main
difference
being
the
absence
of
the
double
bond
at
C-5
(Table
1)
in
compound
2.
The
structure
of
the
aglycone
moiety
was
recognized
to
be
5
a
-spirost-
25(27)-ene-1
b
,3
b
-diol
by
intensive
1
H
and
13
C
NMR
analysis
(Table
1)
and
by
comparison
with
the
literature
data
(Mimaki
et
al.,
1998).
The
sugar
part
was
shown
to
be
similar
to
that
observed
in
compound
1
by
acid
hydrolysis
and
by
correlations
observed
in
the
1
H–
1
H
COSY,
HMQC
and
HMBC
spectral
data.
However,
the
deshielded
chemical
shifts
observed
for
the
fucopyranosyl
C-4
0
and
H-4
0
(
d
80.7
and
4.44,
respectively)
compared
to
those
of
compound
1
(
d
73.9
and
3.51,
respectively)
indicated
that
the
hydroxyl
group
at
C-4
0
was
acylated.
Since
compound
2
(m/z
802)
has
82
more
atomic
mass
units
than
1
(m/z
720),
the
substituent
at
C-4
0
was
identified
as
SO
3
H,
as
confirmed
by
some
fragments
at
m/z
96
[SO
4
]
2
,
80
[SO
3
]

and
64
[SO
2
]

observed
on
the
ESIMS
(Holcapek
et
al.,
2010;
Kougan
et
al.,
2010).
The
structure
of
this
compound
was
also
examined
by
ESI
tandem
mass
spectrometry
(Fig.
2).
The
MS
2
experiment
of
the
quasimolecular
ion
peak
at
m/z
801
[MH]

gave
a
fragment
at
m/z
655
[MH-146]

,
corresponding
to
the
loss
of
the
terminal
rhamnose.
The
MS
3
yielded
an
intense
peak
m/z
225,
attributable
to
the
sulfated
dehydrofucopyranosyl
fragment.
Based
on
the
above
findings,
compound
2
was
thus
elucidated
as
5
a
-spirost-25(27)-ene-1
b
,3
b
-diol-1-O-
a
-
L
-rham-
nopyranosyl-(1!2)-(4-O-sulfo)-
b
-
D
-fucopyranoside,
a
new
ste-
roidal
saponin
to
which
the
trivial
name
fruticoside
I
was
given.
Compound
3
was
isolated
as
an
amorphous
white
powder
from
EtOAc–MeOH.
Its
molecular
formula
C
39
H
66
O
13
was
deduced
from
the
negative-ion
mode
HRESIMS
which
showed
the
ion
peak
at
m/
z
=
777.4189
[M+Cl]

.
This
was
confirmed
by
the
ESIMS
which
exhibited
the
[M+Cl]

at
m/z
777
and
a
prominent
ion
fragment
at
m/z
433
[M+Cl-162-146-2H
2
O]

attributable
to
the
loss
of
one
hexose,
one
desoxyhexose
and
two
water
molecules.
The
1
H
NMR
spectrum
showed
signals
due
to
two
tertiary
methyl
protons
at
d
1.09
(Me-19)
and
0.90
(Me-18),
three
secondary
methyl
protons
0.92
(Me-26
and
Me-27)
and
0.91
(Me-21),
an
olefinic
proton
at
d
5.55
(br
d,
5.7,
H-6)
characteristic
of
a
steroidal
derivative,
two
anomeric
protons
at
d
4.60
(br
s,
H-1
00
)
and
4.26
(d,
7.1,
H-1
0
)
as
well
Table
1
13
C
and
1
H
NMR
data
of
compounds
1–3
(CD
3
OD):
d
in
ppm,
J
in
Hz.
Position
1
2
3
d
13
C
d
1
H
(mult,
J)
d
13
C
d
1
H
(mult,
J)
d
13
C
d
1
H
(mult,
J)
1
85.2
3.32
m
83.7
3.47
(dd,
11.2,
4.0)
84.1
3.48
(dd,
11.0,
4.2)
2
37.8
1.68
(br
t,
11.5);
2.03
m
37.6
1.67
(br
t,
11.6);
2.10
m
37.4
1.57
m;
2.16
m
3
69.4
3.30
m
69.0
3.50
m
69.2
3.39
m
4
43.6
2.18
(dd,
12.2,
5.9);
2.24
(br
t,
11.8)
39.5
1.30
m;
1.46
m
43.5
2.18
(dd,
12.1,
5.2);
2.27
(br
t,
11.9)
5
139.7
/
44.0
1.00
m
139.9
/
6
126.1
5.55
(br
d,
5.6)
29.7
1.34
m
126.0
5.55
(br
d,
5.7)
7
32.8
1.50
m
33.5
1.65
m
32.7
1.49
m;
1.89
m
8
34.2
1.52
m
37.8
1.54
m
34.3
1.42
m
9
51.7
1.23
m
56.3
0.86
m
51.4
1.24
m
10
43.5
/
42.3
/
43.4
/
11
24.8
2.56
m
24.3
2.77
(dd,
3.5,
13.9)
24.7
1.41
m;
2.49
m
12
41.3
1.65
m
41.3
1.70
m
41.5
1.19
m;
1.90
m
13
41.5
/
41.5
/
42.9
/
14
58.3
1.10
m
58.1
1.13
m
56.2
0.95
m
15
33.0
1.29
m,
1.97
m
33.0
1.27
m,
1.95
m
36.6
1.36
m;
2.20
m
16
82.6
4.42
(q,
7.2)
82.6
4.43
m
83.7
4.11
(td,
7.7,
4.3)
17
64.3
1.70
m
64.3
1.70
m
58.8
1.56
m
18
17.3
0.83
s
17.2
0.81
s
13.9
0.90
s
19
15.4
1.08
s
8.9
0.93
s
15.0
1.09
s
20
43.0
1.91
m
43.0
1.92
m
36.7
1.91
m
21
15.0
0.95
(d,
7.0)
15.0
0.95
(d,
7.0)
11.8
0.91
(d,
7.2)
22
110.8
/
110.8
/
74.1
3.49
m
23
34.1
1.70
m
34.1
1.70
m
34.7
1.30
m;
1.56
m
24
29.6
2.22
m,
2.52
m
29.6
2.24
m,
2.52
m
37.3
1.19
m
25
145.3
/
145.4
/
29.8
1.55
m
26
65.9
3.81
(br
d,
11.6);
4.26
(br
d,
11.6)
65.9
3.81
(br
d,
11.6);
4.26
(br
d,
11.6)
23.2
0.92
o
27
109.1
4.73
br
s;
4.76
br
s
109.0
4.73
br
s;
4.77
br
s
23.2
0.92
o
Fuc-1
0
101.0
4.26
(d,
7.7)
100.1
4.36
(d,
7.6)
2
0
75.1
3.62
o
75.8
3.64
o
3
0
77.0
3.59
(dd,
3.3,
8.0)
76.6
3.74
m
4
0
73.9
3.51
o
80.7
4.44
o
5
0
71.6
3.52
o
70.8
3.69
o
6
0
17.0
1.24
(d,
6.3)
17.2
1.29
(d,
6.3)
Rha-1
00
101.6
5.29
(br
d,
1.8)
101.6
5.34
(br
d,
1.8)
105.0
4.60
(br
s)
2
00
72.5
3.86
(dd,
1.8,
3.5)
72.4
3.87
m
73.0
3.71
(br
d,
3.2)
3
00
72.2
3.68
(dd,
3.5,
8.5)
72.1
3.67
o
72.7
3.56
(dd,
9.4,
3.2,
9.4)
4
00
74.3
3.38
(dd,
8.5,
9.6)
74.3
3.38
(dd,
6.4,
9.6)
74.1
3.38
(br
t,
9.4)
5
00
69.6
4.11
m
69.6
4.08
m
71.1
3.65
m
6
00
18.5
1.23
(d,
6.2)
18.1
1.24
(d,
6.0)
17.9
1.23
(d,
6.3)
Gal-1
0
102.1
4.26
(d,
7.1)
2
0
72.7
3.47
o
3
0
75.2
3.45
o
4
0
70.2
3.83
(dd,
8.8,
3.5)
5
0
76.4
3.42
o
6
0
a,b
62.6
3.70
o
o:
overlapped
signals.
R.T.
Fouedjou
et
al.
/
Phytochemistry
Letters
7
(2014)
62–68
64

as
the
methyl
group
of
a
6-deoxyhexopyranosyl
moiety
at
d
1.23
(J
=
6.3,
Me-Rha)
(Table
1).
The
13
C
NMR
spectrum
showed
39
signals,
supporting
the
molecular
formula
deduced
from
the
mass
analysis.
The
salient
feature
of
this
spectrum
was
the
presence
of
a
double
bond
characterized
by
signals
at
d
139.9
(C-5)
and
126.0
(C-
6),
and
two
anomeric
carbons
at
d
105.0
(C-1
00
)
and
102.1
(C-1
0
).
Intensive
interpretation
of
the
1
H
and
13
C
NMR,
1
H–
1
H
COSY,
HSQC
and
HMBC
spectra
(Table
1)
and
comparison
with
published
data
led
to
the
identification
of
the
aglycone
part
as
(22S)-cholest-5-
ene-1
b
,3
b
,16
b
,22-tetrol
(Yokosuka
et
al.,
2012;
Zhang
et
al.,
2007;
Kuroda
et
al.,
2004;
Mimaki
et
al.,
1999,
2001).
Analysis
of
NMR
data
also
allowed
unambiguous
assignments
of
signals
of
sugars
units
and
their
identification
as
a
-rhamnose
and
b
-galactose.
The
coupling
constants
observed
for
the
anomeric
protons
suggested
that
the
anomeric
configurations
of
rhamnose
and
galactose
were
of
the
a
and
b
forms,
respectively
(Agrawal,
1992).
On
the
HMBC
spectrum,
the
cross-peak
correlation
observed
between
the
anomeric
proton
at
d
4.26
(H-1
0
)
and
the
carbon
at
d
84.1
(C-1)
showed
that
the
galactopyranosyl
unit
was
linked
at
C-1
of
the
aglycone.
Finally,
the
correlation
observed
between
the
signal
at
d
4.60
(H-1
00
)
and
the
carbon
at
d
83.7
(C-16)
showed
that
the
rhamnopyranosyl
unit
was
located
at
C-16.
Compound
1
yielded
rhamnose
and
galactose
upon
hydrolysis
which
were
identified
by
comparison
on
TLC
with
authentic
samples.
The
absolute
configuration
of
sugar
residues
was
determined
by
GC
analysis
of
their
chiral
derivatives.
Thus
the
structure
of
compound
3
was
elucidated
as
(22S)-cholest-5-ene-1
b
,3
b
,16
b
,22-tetrol
1-O-
b
-
galactopyranosyl-16-O-
a
-
L
-rhamnopyranoside
a
new
cholestane
bisdesmoside,
trivially
named
fruticoside
J.
2.2.
Biological
activity
Steroidal
saponins
are
reported
to
possess
a
wide
range
of
biological
actions,
including
cytotoxic,
antimicrobial
and
antifun-
gal
activity
(Sautour
et
al.,
2007).
Thus,
the
new
saponins
isolated
from
C.
fruticosa
were
investigated
for
their
cytotoxic
ant
antimicrobial
activity.
The
effect
of
compounds
1–3
on
the
viability
of
MDA-MB
231
human
breast
adenocarcinoma
cell
line,
HCT
116
human
colon
carcinoma
cell
line,
and
A375
human
malignant
melanoma
cell
line
was
evaluated
in
vitro
using
the
MTT
assay.
Compounds
1
and
2
showed
antiproliferative
activity.
IC
50
values
ranged
from
37.83
m
M
to
69.68
m
M
for
1
on
A375
and
MDA-MB
231cells,
respectively,
and
from
46.59
m
M
to
59.97
m
M
for
2
on
A375
and
HCT116
cells,
respectively
(Table
2).
Compound
3
resulted
inactive
on
the
three
tumour
cell
lines
tested.
The
cytotoxic
activity
of
compounds
1
and
2
resulted
moderate
in
comparison
with
that
of
spirostane-type
saponins
isolated
from
Dioscoreaceae
tested
on
A375-S2,
where
the
GI
50
values
ranged
from
2.2
to
3.4
m
M
for
dioscin
and
gracillin
(Sautour
et
al.,
2007).
Same
order
of
magnitude
is
reported
for
gracillin
on
breast
cancer
cell
lines
(GI
50
<
2.44
m
M)
(Hu
and
Yao,
2003).
The
results
on
the
antimicrobial
activity
against
S.
aureus,
E.
coli,
P.
aeruginosa,
E.
faecalis,
and
C.
albicans
are
summarized
in
Table
3.
All
the
isolated
saponins
were
not
able
to
inhibit
the
growth
at
the
highest
concentration
used
in
the
test
(256
mg/L),
with
the
exception
of
compound
2
against
E.
faecalis.
In
this
case
the
MIC
value
was
128
mg/L,
which,
albeit
still
high,
is
indicative
of
a
specific
antibacterial
activity.
The
minor
structural
differences
between
1
and
2
suggested
that
the
presence
of
SO
3
H
group
at
C-4
0
and
the
absence
of
the
double
bond
at
C-5
are
able
to
significantly
decrease
the
MIC
value
of
the
molecules
against
the
enterococcal
species.
Saponins
of
the
diosgenin
series
proved
to
be
inhibitors
of
the
Gram-positive
species
Streptococcus
pneumoniae,
but
were
ineffective
against
E.
faecalis
and
S.
aureus
(Pettit
et
al.,
2005).
These
findings
are
quite
interesting
in
that
the
antibacterial
activity
of
specific
and
well
defined
saponins
is
strictly
and
strongly
related
to
their
fine
structure.
Even
a
single
hydroxyl
group
could
impair
the
activity
(Pettit
et
al.,
2005).
Certain
Fig.
2.
MS
n
spectra
of
compound
2;
(a)
full
mass
spectrum,
(b)
MS
2
spectrum
on
m/z
801
and
(c)
MS
3
spectrum
of
m/z
655.
Table
2
In
vitro
cytotoxic
activity
of
compounds
1–3.
Compounds
Cell
lines
(IC
50
m
M)
MDA-MB
231
a
A375
b
HCT116
c
1
69.68
37.83
39.80
95%
C.I.
64.96–74.74
35.51–40.29
36.65–43.23
2
50.45
46.59
59.97
95%
C.I.
48.59–52.37
43.52–49.88
48.88–73.58
3
>200
>200
>200
95%
C.I.
Cisplatin
7.28
0.62
4.97
95%
C.I.
6.15–7.98
0.48–0.73
4.36–5.65
IC
50
=
the
concentration
of
compound
that
affords
a
50%
reduction
in
cell
growth
(after
72
h
of
incubation).
C.I.
=
Confidence
Interval.
a
Human
breast
adenocarcinoma
cell
line.
b
Human
malignant
melanoma
cell
line.
c
Human
colon
carcinoma
cell
line.
R.T.
Fouedjou
et
al.
/
Phytochemistry
Letters
7
(2014)
62–68
65

saponins,
as
dioscin,
dioscin-related,
diosgenin-related,
and
astropuroside-related
compounds,
have
been
described
as
having
good
antimicotic
activity
(MIC
values
against
C.
albicans,
2–25
mg/
L)
(Pettit
et
al.,
2005;
Zhang
et
al.,
2006;
Sautour
et
al.,
2007).
The
lack
of
activity
shown
by
compounds
1–3
may
be
well
accounted
for
by
the
substantial
differences
in
both
the
composition
and
localization
of
the
sugar
moieties
and
the
structure
of
the
aglycones,
which
are
essential
for
the
antimicrobial
activity
(Pettit
et
al.,
2005).
More
similar
compounds
have
been
recently
described
(Xu
et
al.,
2010),
but
still,
their
structures
are
different
either
in
the
aglycone
and,
even
more,
in
the
sugars
at
C1,
which
were
addressed
as
important
determinants
of
SAR
(Xu
et
al.,
2010).
2.3.
Conclusions
Three
new
steroidal
glycosides
were
isolated
from
the
leaves
of
C.
fruticosa
and
their
structures
elucidated
mainly
by
NMR
data.
Compounds
1
and
2
showed
a
moderate
cytotoxic
activity
against
three
human
cell
lines.
Compound
2
also
showed
a
specific
activity
against
E.
faecalis,
highlighting
how
saponins
deserve
more
attention
as
antibacterials.
This
latter
aspect
is
often
assumed
as
less
important
over
the
antifungal
activity.
Given
the
overwhelm-
ing
natural
diversity
within
the
family
of
saponins,
molecules
with
antimicrobial
activity
could
be
present
in
natural
products
that
might
represent
new
and
effective
antibacterial
agents.
3.
Experimental
3.1.
General
experimental
procedures
Optical
rotations
were
measured
on
PerkinElmer
241
MC
polarimeter.
IR
spectra
were
measured
as
a
film
on
a
KBr
pellet
using
a
FTIR-8400S
Shimadzu
spectrometer.
ESIMS
was
carried
out
on
an
Agilent
Technologies
LC/MSD
Trap
SL
(G2445D
SL).
ESIMS
n
was
performed
in
FIA
(Flow
Injection
Analysis)
using
MeOH
as
solvent
(5
m
L/min,
nebulizer
pressure
15
psi),
drying
gas
flow
4
L/
min,
drying
gas
temperature
325
8C.
HRESIMS
were
recorded
on
an
Agilent
Thecnologies
6540
UHD
Accurate
Mass
Q-ToF
LC/MS.
NMR
spectra
were
performed
in
CD
3
OD
on
a
varian
Mercury
plus
Spectrometer
(400
MHz
for
1
H
and
100
MHz
for
13
C).
All
chemical
shifts
(
d
)
are
given
in
ppm
units
with
reference
to
the
residual
solvent
signal
and
the
coupling
constants
(J)
are
in
Hz.
Column
chromatography
was
performed
using
Sephadex
LH-20
and
silica
gel
60
(0.040–0.063
mm,
Merck).
TLC
was
carried
out
on
precoated
Kiegel
60
F
254
(Merck)
plates
developed
with
EtOAc–MeOH–H
2
O
(90–10–5,
95–5–2,
97–3–1)
and
with
EtOAc–MeOH
98–2.
TLC
plates
were
visualized
under
UV
light
(254
and
365
nm)
or
by
spraying
with
50%
aqueous
H
2
SO
4
and
heating
for
10
min
at
110
8C.
3.2.
Plant
material
The
leaves
of
C.
fruticosa
were
collected
in
Dschang
(West
region
of
Cameroon)
in
December
2011.
The
plant
was
identified
by
Mr
J.P.
Dondjang,
botanist
at
the
Department
of
Forestry,
University
of
Dschang,
where
a
voucher
specimen
(Ref:
LACAPE0001)
was
deposited.
3.3.
Extraction
and
isolation
The
dried
and
pulverized
leaves
(3
kg)
were
extracted
three
times
(each
time
for
24
h)
with
MeOH.
The
combined
filtrate
was
concentrated
under
reduced
pressure
to
give
a
dark
residue
(503
g),
which
was
suspended
in
distilled
water
and
partitioned
successively
with
n-hexane,
EtOAc
and
n-butanol,
yielding
58
g,
69
g,
and
60
g
of
dry
extracts,
respectively.
Part
of
the
EtOAc
extract
(59
g)
was
fractionated
on
a
silica
gel
column
chromatography
using
a
gradient
of
EtOAc
in
n-hexane
and
then
a
gradient
of
MeOH
in
EtOAc,
to
give
ten
main
fractions
(A–J).
Fraction
C
(0.5
g)
(eluted
with
Hexane-EtOAc
40%)
was
chromatographed
on
a
silica
gel
column
with
hexane–EtOAc
30%
as
eluent
to
yield
farrerol
(27
mg).
Fraction
F
(7.12
g)
(eluted
with
EtOAc)
was
submitted
to
silica
gel
CC
eluting
with
EtOAc–MeOH
(98–2)
and
on
Sephadex
LH-20
using
MeOH
as
to
give
quercetin
3-O-[6-trans-p-coumaroyl]-
b
-
D
-gluco-
pyranoside
(7
mg).
Recrystallization
of
fraction
G
(2.6
g)
(eluted
with
EtOAc)
yielded
apigenin
8-C-
b
-
D
-glucopyranoside
(57
mg)
and
the
resulting
filtrate
was
chromatographed
on
silica
gel
eluting
with
EtOAc–MeOH–H
2
O
(97–3–1)
then
on
Sephadex
LH-20
column
chromatography
(eluted
with
MeOH)
to
afford
quercetin
3-O-
b
-
D
-glucopyranoside
(12
mg).
Fraction
H
(8.3
g)
(EtOAc–
MeOH
5%)
was
rechromatographed
on
a
silica
gel
column
using
EtOAc–MeOH
(98–2)
as
eluent.
Evaporation
of
the
solvent
followed
by
further
crystallization
afforded
compound
1
(22
mg).
Fraction
J
(3.5
g)
(eluted
with
EtOAc–MeOH
10%)
was
purified
on
a
silica
gel
column
with
EtOAc–MeOH–H
2
O
(90–10–5)
as
eluent
and
afforded
a
sub-fraction
containing
a
major
compound,
which
was
further
chromatographed
on
silica
gel
column
using
EtOAc–MeOH–H
2
O
(90–10–5)
to
yield
compound
2
(10
mg).
Fraction
I
(9.5
g)
(EtOAc-
MeOH
10%)
was
repeatedly
chromatographed
on
silica
gel
with
EtOAc–MeOH–H
2
O
(95–5–2)
as
eluent
to
afford
compound
3
(29
mg).
Part
of
the
n-butanol
extract
(50
g)
was
dissolved
in
MeOH,
fixed
on
silica
gel
and
then
fractionated
by
column
chromatography
eluted
with
EtOAc
and
increasing
amounts
of
MeOH.
The
fraction
obtained
by
eluting
with
EtOAc–MeOH
(75–
25)
(9.04
g)
was
rechromatographed
on
a
silica
gel
column
using
EtOAc–MeOH
(90–5)
as
eluent
to
afford
6
sub-fractions.
One
of
the
above
sub-fractions
(157
mg)
was
purified
on
a
Sephadex
LH-20
column
eluted
with
MeOH
to
yield
quercetin
3-rutinoside
(57
mg).
3.3.1.
Fruticoside
H
(1)
White
amorphous
powder;
½a
25
D
¼
101:5
(c
0.2,
MeOH);
IR
(KBr)
y
max
3300
(OH),
2981
and
2941
(CH),
1446
(C55C),
1062
(C–O)
cm
1
;
for
1
H
and
13
C
spectroscopic
data,
see
Table
1;
HRESIMS:
m/z
743,3986
[M+Na]
+
(calcd.
for
C
39
H
60
O
12
Na,
743.3982);
ESIMS:
m/z
743
[M+Na]
+
.
3.3.2.
Fruticoside
I
(2)
White
amorphous
powder;
½a
25
D
¼
50:5
(c
0.6,
MeOH);
IR
(KBr)
y
max
3433
(OH),
2904
(CH),
1456
(C55C),
1224
(S–O
stretching),
1056
(C–O)
cm
1
;
for
1
H
and
13
C
spectroscopic
data,
see
Table
1;
HRESIMS:
m/z
801.3727
[MH]

(calcd.
for
C
39
H
61
O
15
S,
801.3731);
ESIMS:
m/z
801
[MH]

;
Tandem
MS/
MS:
m/z
801
[MH]

,
655
[MH-146]

,
225
m/z
[MH-146-
aglycone]

.
3.3.3.
Fruticoside
J
(3)
White
amorphous
powder;
½a
25
D
¼
53:0
(c
0.2,
MeOH);
IR
(KBr)
y
max
3420
(OH),
2924
(CH),
1452
(C55C),
1059
(C–O)
cm
1
;
1
H
and
13
C
spectroscopic
data,
see
Table
1;
HRESIMS:
m/z
777,4189
[M+Cl]

(calcd.
for
C
39
H
66
O
13
Cl,
777.4192);
ESIMS:
m/z
777
[MCl]

,
433
[MCl-162-146-2H
2
O]

.
Table
3
In
vitro
antimicrobial
activity
of
compounds
1–3.
Compounds
MIC
(mg/L)
S.
aureus
E.
faecalis
E.
coli
P.
aeruginosa
C.
albicans
1
>256
>256
>256
>256
>256
2
>256
128
>256
>256
>256
3
>256
>256
>256
>256
>256
MIC
=
the
minimal
inhibitory
concentration
is
the
concentration
at
which
there
is
no
visible
microbial
growth
after
18–24
h
of
incubation
(24–48
h
for
C.
albicans).
R.T.
Fouedjou
et
al.
/
Phytochemistry
Letters
7
(2014)
62–68
66

3.4.
Acid
hydrolysis
A
solution
of
each
compound
(3
mg)
in
water
(1
ml)
and
2
N
aqueous
CF
3
COOH
(10
ml)
was
refluxed
at
100
8C
on
water
bath
for
2
h.
The
mixture
was
then
diluted
in
water
(10
ml)
and
extracted
with
EtOAc
(3
4
ml).
The
combined
EtOAc
layers
were
washed
with
H
2
O
and
evaporated
to
dryness
to
afford
the
artefactual
aglycones.
The
aqueous
residue
was
concentrated
to
dryness
by
adding
MeOH
to
remove
acid
and
analyzed
by
TLC
in
comparison
with
standards
D
-fucose,
L
-rhamnose
and
D
-galac-
tose.
The
absolute
configuration
of
sugar
residues
was
deter-
mined
by
GC
analysis
(Hara
et
al.,
1987)
of
their
chiral
derivatives
(Elbandy
et
al.,
2003).
3.5.
MTT
cytotoxicity
assay
MDA-MB
231
cells
(human
breast
adenocarcinoma
cell
lin),
and
A375
cells
(human
malignant
melanoma
cell
line)
were
cultured
in
Dulbecco’s
Modified
Eagle’s
Medium
(DMEM)
with
2
mM
L
-glutamine,
100
IU/mL
penicillin,
100
m
g/mL
streptomy-
cin,
and
supplemented
with
10%
heat
inactivated
foetal
bovine
serum
(HI-FBS)
(PAA
Laboratories
GmbH,
Austria,
UE).
HCT116
cells
(human
colon
carcinoma
cell
line)
were
cultured
in
RPMI1640
medium
with
2
mM
L
-glutamine,
100
IU/mL
penicil-
lin,
100
m
g/mL
streptomycin,
and
supplemented
with
10%
HI-
FBS.
Cells
were
cultured
in
a
humidified
atmosphere
at
378
C
in
presence
of
5%
CO
2
.
The
MTT
assay
was
used
as
a
relative
measure
of
cell
viability.
Cell
viability
assays
were
carried
out
as
described
(Quassinti
et
al.,
2012).
Briefly,
cells
were
seeded
at
the
density
of
2

10
4
cells/ml.
After
24
h,
samples
were
exposed
to
different
concentrations
of
compound
(6.25–200
m
M).
Cells
were
incu-
bated
for
72
h
in
a
humidified
atmosphere
of
5%
CO
2
at
37
8C.
At
the
end
of
incubation,
each
well
received
10
m
l
of
3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide
(MTT)
(5
mg/ml
in
phosphate-buffered
saline,
PBS)
and
the
plates
were
incubated
for
4
h
at
37
8C.
The
extent
of
MTT
reduction
was
measured
spectrophotometrically
at
540
nm
using
a
Titertek
Multiscan
microElisa
(Labsystems,
Helsinki,
Finland).
Experiments
were
conducted
in
triplicate.
Cytotoxicity
is
expressed
as
the
concentration
of
compound
inhibiting
cell
growth
by
50%
(IC
50
).
The
IC
50
values
were
determined
using
GraphPad
Prism
4
computer
programme
(GraphPad
Software,
S.
Diego,
CA,
USA).
3.6.
Antimicrobial
activity
test
Compounds
1–3
were
tested
against
a
panel
of
microorganisms
including
S.
aureus
ATCC
25923,
E.
coli
ATCC
25922,
P.
aeruginosa
ATCC
27853,
E.
faecalis
ATCC
29212,
and
C.
albicans
ATCC
24433.
Bacterial
strains
were
cultured
overnight
at
37
8C
in
blood
agar
plates
with
the
exception
of
Candida
that
was
grown
in
RPMI1640.
Antibiotic
susceptibility
testing
was
performed
by
the
micro-
dilution
method,
following
the
international
guidelines
of
the
CLSI
(CLSI,
2009).
Briefly,
a
10
mg/mL
stock
solution
for
each
of
the
compounds
to
be
tested
was
prepared
in
MeOH.
Then,
twofold
serial
dilutions
of
each
compound
were
prepared
in
96-well
plates,
starting
from
256
mg/L,
in
Cation
Adjusted
Mueller
Hinton
Broth
(RPMI1640
for
C.
albicans).
An
equal
volume
of
the
microbial
inoculum
(10
6
cfu/mL),
obtained
by
direct
colony
suspension
of
an
overnight
culture,
was
added
to
each
well
of
the
microtiter
plate
containing
0.1
mL
of
the
serially
diluted
test
molecule.
After
incubation
for
18–24
h
at
35
8C
(24–48
h
in
the
case
of
Candida),
in
normal
atmosphere,
Minimum
Inhibitory
Concentrations
(MICs)
were
defined
as
the
lowest
concentration
of
compound
able
to
inhibit
the
growth
of
the
microorganisms.
All
tests
were
done
in
duplicate.
Acknowledgements
Dr
R.B.
Teponno
is
grateful
to
the
Academy
of
Sciences
for
the
Developing
World
(TWAS)
for
financial
support
(grant
RGA
No.
09-
152
RG/CHE/AF/AC
I-UNESCO
FR:3240230339).
The
authors
are
also
grateful
to
the
Italian
Ministry
of
Education
(MIUR)
for
supporting
this
research
through
the
COOPERLINK
2011
(Prot.
CII113PPUC
‘‘Tesi
di
Dottorato
sullo
studio
di
molecole
biologica-
mente
attive
estratte
da
piante
della
medicina
tradizionale
del
Camerun’’.
Project
Manager
L.
Barboni).
Appendix
A.
Supplementary
data
Supplementary
data
associated
with
this
article
can
be
found,
in
the
online
version,
at
http://dx.doi.org/10.1016/j.phytol.2013.
10.001.
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