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Nguyen P-D et al. Antimicrobial Constituents from Leaves … Planta Med Int Open 2018; 5: e14–e23
Original Papers
Introduction
Dolichandrone spathacea (L.f.) Seem. (Bignoniaceae) also known as
mangrove trumpet tree, is a common tree growing wild in river
banks and mangroves of the Asia-Pacic area [1]. The leaves are used
as an antitumor, antiseptic, and to treat oral thrush (as mouthwash),
nervous diseases and atulence in many countries of Southeast Asia
[1, 2]. The juice of the leaves is used orally against bronchitis in India
[3]. In Vietnam, this plant is part of a traditional medication (Tiêu
Phong Nhuân Gan), used against hepatic disorders, skin diseases,
allergies, and as detoxier, anti-inammatory and laxative. Several
biological activities of leaves extracts were measured showing for
example, that the polar extracts possessed a high antiradical activ-
ity [2] and that the aqueous methanol extract exhibited an inhibi-
tory eect against rat intestinal maltase [4]. Literature also reports
that a methanol extract from the leaves exhibited a strong growth
inhibition of six strains of methicillin-resistant Staphylococcus aureus
(MRSA) [5]. The same article demonstrated, in a preliminary ap-
proach, the detection of avonoids, saponins, triterpenes, and tan-
nins in the leaves but without any structural characterization [5]. In
the present paper, we report the isolation and characterization of
Nguyen Phuc-Dam et al. Antimicrobial Constituents from Leaves … Planta Med Int Open 2017; 00: 00–00
Antimicrobial Constituents from Leaves of Dolichandrone spathacea
and Their Relevance to Traditional Use
Authors
Phuc-Dam Nguyen1, Amin Abedini1, 2, Sophie C. Ganglo2, Catherine Lavaud1
Aliations
1 Institut de Chimie Moléculaire de Reims, CNRS UMR
7312, Reims, France
2 Laboratoire de Microbiologie, EA 4691, UFR de
Pharmacie, Reims, France
Keywords
Dolichandrone spathacea, Bignoniaceae, iridoid glycosides,
triterpenoid saponins, antibacterial activity
received 14.07.2017
revised 14.11.2017
accepted 11.12.2017
Bibliography
DOI https://doi.org/10.1055/s-0043-125339
Planta Med Int Open 2018; 5: e14–e23
© Georg Thieme Verlag KG Stuttgart · New York
ISSN 2509-9264
Correspondence
Prof. Catherine Lavaud
Institut de Chimie Moléculaire de Reims (ICMR)
CNRS UMR 7312
BP 1039
51687 Reims Cedex 2
France
Tel.: + 33/3/26 91 31 39
catherine.lavaud@univ-reims.fr
Supporting information is available online at
http://www.thieme-connect.de/products
ABSTRACT
Five new compounds, three iridoid glycosides (1-3) and two
triterpenoid saponins (4, 5), along with thirty-two known com-
pounds were isolated from the methanolic extract of the leaves
of Dolichandrone spathacea. This traditional medicinal plant is
widely used in Asia and India as antiseptic, for bronchitis and
thrush treatment, and the methanolic extract has been shown
to possess antibacterial activity against methicillin-resistant
Staphylococcus aureus. The new iridoids were esteried deriva-
tives of 6-ajugol and 6-catalpol, and the new saponins were
glucosides of two polyhydroxy triterpenes with ursan skeleton.
Their structures were elucidated by spectroscopic methods,
including 1D and 2D NMR experiments and HR-ESI-MS analysis,
and from comparison with the literature. This study aimed at
investigating extracts and isolated compounds for their anti-
microbial activities against bacterial and yeast strains, in order
to validate the uses of the plant in folk medicine. The 6-O-es-
teried iridoids had weaker antibacterial activity; verbascoside
and p-methoxycinnamic acid, the major compounds of the
methanol extract, possessed strong antibacterial activity,
which could account for the traditional antiseptic and anti-in-
fectious uses of the leaves of D. spathacea.
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Nguyen P-D et al. Antimicrobial Constituents from Leaves … Planta Med Int Open 2018; 5: e14–e23
phytochemical constituents from leaves of D. spathacea, and the
evaluation of antimicrobial activities of extracts and isolated com-
pounds against dierent microorganisms.
Results and Discussion
The leaves of D. spathacea were extracted by successive percola-
tions with petroleum ether, chloroform, ethyl acetate, methanol,
and 80 % aqueous methanol. The methanolic extract was puried
by using a combination of liquid chromatographic techniques to
yield 37 compounds. Among these, ve were new compounds:
three iridoid glucosides (1-3) and two triterpenoid saponins (4 and
5) (▶Fig. 1). The 32 known isolated compounds were: 6-O-p-E-
coumaroyl-ajugol [6], 6-O-E-caeoyl-ajugol and 6-O-E-isoferuloyl-
ajugol [7], nemorososide [8], 6-O-(E)-cinnamoyl-catalpol [9], spe-
cioside [10], 6-(E)-p-methoxycinnamoyl-catalpol [11], a mixture
of (E)- and (Z)-6-p-methoxycinnamoyl-catalpol [12], verminoside
and minecoside [13], nemoroside and 6’’(Z)-nemoroside [14], ix-
oside [15], arjunglucoside I [16], decaeoylacteoside [17], verbas-
coside (or acteoside) [18], isoverbascoside (or isoacteoside) [19],
luteolin, luteolin-7-O-β-D-glucopyranoside, luteolin-7-O-β-D-
glucuronide and luteolin-7-O-rutinoside [20–23], (2E,6E)-8-hy-
droxy-2,6-dimethyl-2,6-octadienoic acid [24], (2E,6Z)-8-hy-
droxy-2,6-dimethyl-2,6-octadienoic acid [25], 6 R- and 6 S-(2E)-
8-hydroxy-2,6-dimethyl-2-octenoic acid in mixture [26, 27],
p-hydroxybenzoic acid [28], vanillic acid [29], p-hydroxycinnamic
and p-methoxycinnamic acids [30], isoferulic acid [31], and 6 S,9 S-
roseoside [32, 33].
The new compounds (1-5) possessed the same sugar residue in
their structures (▶Table 1 and 2), which was determined as D-glu-
cose after acid hydrolysis and chiral HPLC analysis. NMR showed
that it was in the β-D-pyranosyl conguration.
The 1H-NMR data (▶Table 1) of 1 and 2 revealed the character-
istic signals of an ajugol part [6] with an acetal proton at δH 5.53,
two cis olenic protons at δH 6.24 and 5.00, one methyl singlet at
δH 1.41, and a deshielded hydroxymethine proton at δH 4.96. The
13C-NMR (DEPT and HSQC) data conrmed the presence of the aju-
gol moiety (▶Table 1) [6], and the relative stereochemistry was
confirmed by observation of ROE effects between H-5β/H-9β,
H-1α/H-10α, H-10α/H-7α, H-7α/H-6α, and the absence of an ROE
eect between H-5β/H-1α and H-6α /H-9β.
The molecular formula of 1 was determined to be C25H32O11 by
HR-ESI-MS analysis [molecular ion at m/z 531.1848 [M + Na] +
(calcd. for C25H32O11Na, 531.1842)]. By subtracting the ajugol part,
there remained a C10H24O9 (161 uma) residue. It was assigned to a
p-O-methoxycinnamoyl group by NMR: AA’BB’ system of four aro-
matic protons at δH 7.56 (2 H) and 6.97 (2 H), one E-double bond
at δH 7.68 and 6.43 (J = 16.0 Hz), and one methoxy group at δH 3.85.
The ester carbonyl carbon at δC 167.4 (C-9’’) showed an HMBC cor-
relation with H-6 of ajugol moiety and therefore, compound 1 was
determined as 6-O-(p-methoxy-E-cinnamoyl)-ajugol.
Iridoid glucoside (2) showed a molecular ion peak [M + Na] + at
m/z 539.2462 (calcd. for C 25H40O11Na, 539.2468) in agreement
with a C 25H40O11 formula. Its 1H- and 13C-NMR spectra (▶Table 1)
were similar to those of 1 with characteristic signals for a 6-esteri-
ed derivative of ajugol. The 13C-NMR and DEPT spectra of 2 exhib-
ited ten carbons for a monoterpenic acid elucidated as 8-hy-
droxy-2,6-dimethyl-2-octenoic acid which was isolated as pure
known compound (see below) [26, 27] (▶Table 1). An E-congu-
ration could be assigned to the trisubstituted double bond accord-
ing to the shielding of CH3-9’’ at δC 12.4 in comparison with δC
22-25 for Z-conguration [34]. To determine the absolute cong-
uration of the CH-6’’, we carried out the alkaline hydrolysis of 2 and
then the monoterpenic acid was puried by semi-preparative HPLC
[18]. The positive sign of the optical rotation of the isolated acid
indicated that it was 6 R-(2E)-8-hydroxy-2,6-dimethyl-2-octenoic
acid. The absence of any supplementary peak in the 13C as well as
1H NMR spectra demonstrated that 2 was diastereoisomerically
pure despite the fact that the optical rotation value of the acid
([α]D = + 3.7) was lower than those found in the literature [26, 27].
This discrepancy is most probably due to diculties in measuring
rotations on tiny amounts of material. We concluded that 2 was
6’’R-O-(2E) -8-hydroxy-2,6-dimethyl-2-octenoyl-ajugol.
The HR-ESI-MS analysis of the third new iridoid glucoside (3) re-
vealed a molecular formula of C25H38O12 with the molecular ion at
m/z 553.2256 [M + Na] + (calcd. for C25H38O12Na, 553.2261). The
comparison of 1H- and 13C-NMR spectral data (▶Table 1) of com-
pounds 2 and 3 showed great similarities for the ester monoter-
pene parts. The iridoid skeleton of 3 was identied as catalpol [14]
with only one deshielded proton H-7 at δH 3.70 (brs) and two oxy-
genated carbons for CH2OH-10 at δC 61.3 and –CHO–7 at δC 60.2
[35]. The relative stereochemistry of the six asymmetric centers
was conrmed by analysis of ROE eects. The stereochemistry of
the carbon C-6'' of the monoterpenic acid was determined by the
same method as that employed for hydrolysis of 2. The same opti-
cal rotation ([α]D = + 3.7) was measured and compound 3 was iden-
tied as 6’’R-O-(2E)-8-hydroxy-2,6-dimethyl-2-octenoyl)-catalpol.
The positive HR-ESI-MS of saponin (4) gave a molecular ion peak
[M + Na] + at m/z 673.3934 (calcd. for C36H58O10Na 673.3928) cor-
responding to a molecular formula C36H58O10. The presence of 36
carbons was observed in the 13C-NMR spectrum including 6 car-
bons for a β-D-glucopyranose and 30 carbons for a Δ12 ursene skel-
eton with δC 130.0 (CH-12) and 138.9 (C-13; ▶Table 2). The glu-
cose unit was linked to the triterpene moiety at position C-28 since
an HMBC correlation was observed between the anomeric proton
H-1’ and the carbonyl C-28 at δC 178.5 [36]. The aglycone was de-
termined to be triterpene uncaric acid (or 3β,6β,19α-trihydroxyurs-
12-en-28-oic acid) [37]. The 3β–OH conguration was conrmed
by the large coupling constant 3JH-3ax/H-2ax = 11.7 Hz suitable with
an (α) axial H-3, the hydroxyl group being in equatorial position (β).
The ROE eects observed between H-6 and H-5α, and between
H-6 and H-23α indicated a 6β–OH conguration. The third hydrox-
yl group was localized in axial position (19α-OH) due to the obser-
vation of ROE between the axial proton H-18β and the equatorial
methyl CH3-29β. Thus, compound 4 was identied as 28-O-β-D-
glucopyranosyl-3β, 6β, 19α-trihydroxyurs-12-en-28-oic acid,
named 28-O-β-D-glucopyranosyl uncaric acid.
The positive HR-ESI-MS of saponin (5) displayed a molecular ion
peak [M + Na] + at m/z 689.3869 (calcd. for C 36H58O1Na, 689.3877)
consistent with the molecular formula C36H58O11, therefore a hy-
droxylated derivative of saponin 4. Comparison of the NMR spec-
tral data of 4 and 5 revealed signicant similarities (▶Table 2); the
dierence was the existence of only six methyl groups and an ad-
ditional hydroxyl group located at position C-23 or C-24 according
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Nguyen P-D et al. Antimicrobial Constituents from Leaves … Planta Med Int Open 2018; 5: e14–e23
Original Papers Thieme
to HMBC correlations between the supplementary protons CH2-
OH and carbons C-3, C-4, and C-5. The ROE eects observed be-
tween H-6α/H-5α, and between H-6α and CH2-OH conrmed the
α-equatorial position of 23-CH2-OH. The expected eects due to
24-hydroxylation were well observed on C-23 (Δα = + 38.4), C-4
(Δβ = + 3.5), C-24 (Δγ = -3.5), C-5 (Δγ = -6.7), and C-3 (Δγ = -6.3;
▶Table 2). Thus, the aglycone of 5 was 3β, 6β, 19α, 23-tetrahy-
droxyurs-12-en-28-oic acid [38], and saponin 5 was identied as
RO H
H
HO
O
O
R =
1
R =
RO
O
O
O
O
O
H
HO
HO
OH
OH
OH
H
3
R =
2
1’ 5’
2’ 3’ 4’
6’
OH
OH
OH
HO
O
O
O
HO
1’’
1’’
2’’
2’’
4’’
4’’
3’’
3’’
5’’
5’’
6’’
6’’
7’’
7’’
8’’
10’’
HO
29
12
HO
OH
24 23
R
4R = H
R = OH
27
13
14 15
17
16
11 26
22
18
19 20
21
O
OHO
O
2’ 3’ 4’
5’
1’ 6’
OH
OH
OH
28
5
25
1
2
3456
10 9
8
7
30
8’’
9’’
9’’
OCH3
3
4
7
65
9
1
8
10
▶Fig. 1 Chemical structures of iridoids (1, 2, 3) and saponins (4, 5).
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Nguyen P-D et al. Antimicrobial Constituents from Leaves … Planta Med Int Open 2018; 5: e14–e23
28-O-β-D-glucopyranosyl-3β, 6β, 19α 23-tetrahydroxyurs-12-en-
28-oic acid, or 28-O-β-D-glucopyranosyl-23-hydroxy-uncaric acid.
MIC were determined for the ve extracts prepared from the
leaves of D. spathacea against 8 Gram-positive bacterial strains,
9 Gram-negative bacterial strains, and 5 yeast strains (▶Table 3).
The results demonstrated stronger antibacterial activity of all ex-
tracts against Gram-positive bacteria, and against two pathogenic
bacteria, Streptococcus pyogenes and Shigella sonnei (MIC ≤ 0.3 mg/
mL). The AcOEt extract was the most active against all tested mi-
croorganisms. The antimicrobial activity of the isolated compounds
was rst evaluated with an immersion bioautography method [39]
against Staphylococcus aureus CIP 53.154 (▶Table 4). Five com-
pounds, among them decaeoylacteoside and verbascoside that
showed growth inhibition areas close to the ones observed with
the antibiotic controls, as well as luteolin, p-methoxycinnamic acid
and 6-O-E-caeoyl-ajugol, seemed promising antimicrobial candi-
dates. Our results were in agreement with the literature on anti-
bacterial activity of verbascoside [40], luteolin [41], and p-meth-
oxycinnamic acid [42].
A serial dilution technique in 96-well plates was used to determine
the MIC of the active compounds against ve bacteria (▶Table 4);
the best inhibitory activity was found for the two phenylethanoid di-
glycosidic compounds, decaffeoylacteoside and verbascoside
(MIC = 31 µg/mL), and for p-methoxycinnamic acid (MIC = 62 µg/mL).
In summary, it may be concluded from this study that iridoid
glucosides could be considered as chemical markers of Bignoniace-
ae, represented here by sixteen compounds amongst which three
were never described before. The 6-O-esteried iridoids like mine-
coside and 6-O-caeoyl-ajugol had weaker antibacterial activity
than reported for the non-esteried aucubin [43]. Six compounds
of the methanol extract of D. spathacea showed a strong inhibitory
activity against resistant bacterial strains, particularly against
▶Table 1 1H- and 13C-NMR spectroscopic data (CD3OD) of iridoids (1), (2) and (3).
(1) (2) (3)
δH (J in Hz) δCδH (J in Hz) δCδH (J in Hz) δC
15.53, d (2.5) 92.0 5.56, d (2.4) 93.4 5.18, d (9.6) 95.1
36.24, dd (6.3, 2.2) 139.7 6.23, dd (6.3, 2.2) 141.1 6.39, d (6.0) 142.4
45.00, dd (6.3, 2.6) 103.2 4.97, dd (6.3, 2.7) 104.6 4.98, dd (5.6, 4.1) 102.9
52.95, dd (9.1, 2.3) 38.0 2.90, dq (9.2, 2.2) 39.3 2.61-2.56, m 36.7
64.96, ddd (6.5, 4.1, 2.7) 78.9 4.89, m 80.5 4.98, d (7.7) 81.6
72.02, dd (14.2, 4.0) β
2.27, dd (14.2, 6.4) α
46.5 2.23, dd (14.3, 6.5) α
1.98, dd (14.3, 4.0) β
47.8 3.70, brs 60.2
877.7 79.0 66.8
92.60, dd (9.3, 2.3) 50.2 2.59, dd (9.2, 2.0) 51.6 2.63, dd (9.3, 7.9) 43.2
10 1.41, s 24.7 1.39, s 26.1 4.18, d (13.2)
3.85, d (13.1)
61.3
β-D-glucopyranose (C-1)
1’ 4.69, d (8.0) 98.0 4.68, d (7.9) 99.4 4.81, d (7.9) 99.7
2’ 3.22, dd (9.2, 7.9) 73.4 3.22, dd (9.2, 8.1) 74.8 3.30, dd (8.8, 8.3) 74.9
3’ 3.39, dd (9.2, 8.7) 76.6 3.59, t (8.9) 78.0 3.43, t (9.0) 77.7
4’ 3.31, dd (9.2, 8.6) 71.8 3.29, dd (9.7, 8.5) 71.7 3.28, t (9.3) 71.8
5’ 3.32-3.35, m 76.8 3.32-3.35, m 78.2 3.35, ddd (9.4, 6.3, 1.5) 78.7
6’ 3.68, dd (12.0, 6.0)
3.92, dd (12.0, 2.2)
61.5 3.68, dd (12.0, 5.9)
3.92, dd (12.0, 3.1)
62.9 3.67, dd (12.2, 6.1)
3.95, brd (11.9)
62.9
6-Acyl ester
1’’ 167.4 169.5 169.5
2’’ 6.43, d (16.0) 115.0 128.8 128.3
3’’ 7.68, d (16.0) 144.8 6.83, td (7.5, 1.3) 144.3 6.88, t (7.3) 145.0
4’’ 127.0 2.20-2.32, m 27.2 2.22-2.35, m 27.3
5’’ 7.56, d (8.6) 129.6 1.47-1.54, m
1.29-1.34, m
36.9 1.49-1.56, m
1.30-1.37, m
36.9
6’’ 6.97, d (8.7) 114.0 1.64-1.66, m 30.5 1.60-1.67, m 30.5
7’’ 161.9 1.59-1.64, m
1.35-1.40, m
40.6 1.60-1.65, m
1.41.37-1.43, m
40.6
8’’ 7.56, d (8.6) 114.0 3.62-3.67, m
3.58-3.3.62, m
60.9 3.58-3.66, m 60.9
9’’ 6.97, d (8.7) 129.6 1.85, s 12.4 1.89, s 12.5
10’’ 0.97, d (6.5) 19.8 0.97, d (6.4) 19.8
OCH33.85 (s) 54.5
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Original Papers Thieme
P. aeruginosa and E. faecalis. Dicaeoylacteoside, verbascoside, and
p-methoxycinnamic acid, the major compounds of the methanol
extract, possessed strong antibacterial activity against most of the
strains tested (MIC < 100 µg/mL). This result may explain the use of
this species in folk medicine to treat oral thrush, bronchitis, and eye
infection as well as cutaneous antiseptic. The large number and
wide diversity of isolated compounds from leaves of D. spathacea
supported the other traditional uses of this plant in gastrointesti-
nal diseases as depurative, carminative, and against liver disease.
▶Table 2 1H- and 13C-NMR spectroscopic data (CD3OD) of saponins (4) and (5).
(4) (5)
δH (J in Hz) δCδH (J in Hz) δC
10.98-1.03 m
1.58-1.66, m
42.1 0.94-1.04, m
1.58-1.65, m
41.7
21.58-1.66, m
1.70-1.78, m
28.0 1.58-1.65, m
1.71-1.79, m
27.6
33.10, dd (11.7, 4.1) 80.2 3.58, dd (11.7, 3.8) 73.9
440.7 44.2
50.75, brs 57.2 1.19, brs 49.5
64.50, brs (w1/2 = 7.5) 69.0 4.41, brs (w1/2 = 7.8) 68.9
71.55, dd (14.5, 2.0) eq
1.70-1.78, m ax
41.8 1.51, brd (14.2)
1.78-1.85, m
41.4
840.3 40.3
91.70-1.76, m 49.0 1.71-1.79, m 48.9
10 37.6 37.3
11 2.00-2.12, m 24.7 2.01-2.13, m 24.7
12 5.36, t (3.4) 130.0 5.36, brt (3.4) 130.0
13 138.9 138.9
14 43.1 43.1
15 1.91, td (14.0, 4.4) ax
1.03, m eq
29.7 1.91, td (13.9, 3.9) ax
1.01-1.04, m eq
29.7
16 2.62, td (13.3, 4.3) ax
1.64-1.68, m eq
26.6 2.62, td (13.3, 4.3) ax
1.66, brd (13.0) eq
26.6
17 49.3 48.6
18 2.55, brs 55.0 2.25, s 55.0
19 73.7 73.7
20 1.34-1.40, m 43.0 1.35-1.40, m 43.0
21 1.29-1.24, m
1.73-1.77, m
27.2 1.24-1.29, m
1.71-1.79, m
27.2
22 1.64-1.68, m
1.78-1.83, m
38.3 1.66, brd (12.6)
1.78-1.85, m
38.3
23 1.07, s 28.4 3.50, d (10.9)
3.62, d (11.0)
66.8
24 1.18, s 17.6 1.09, s 14.1
25 1.33, s 17.4 1.35, s 17.7
26 1.07, s 18.7 1.07, s 18.7
27 1.32, s 24.7 1.33, s 24.7
28 178.5 178.5
29 1.23, s 27.1 1.23, s 27.1
30 0.95, d (6.7) 16.6 0.95, d (6.7) 16.6
β-D-glucopyranose (C-28)
1’ 5.33, d (8.1) 95.9 5.33, d (8.2) 95.9
2’ 3.34, brt (8.2) 73.9 3.35, t (8.2) 73.9
3’ 3.43, t (8.9) 78.3 3.43, t (8.8) 78.3
4’ 3.37, t (9.0) 71.2 3.38, dd (9.4, 8.5) 71.2
5’ 3.32-3.36, m 78.6 3.32-3.36, m 78.6
6’ 3.82, dd (11.8, 2.0)
3.70, dd (11.9, 4.6)
62.4 3.83, dd (12.0, 1.9)
3.71, dd (12.0, 4.6)
62.4
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Material and Methods
General experimental procedures
Optical rotations were determined with a Perkin-Elmer 341 pola-
rimeter. UV-Vis spectra were measured with a Shimadzu UV/Vis
U-2450 spectrophotometer. IR spectra were recorded on a Nicolet
Impact 410 FTIR spectrometer. HR-ESI-MS experiments were per-
formed using a Micromass Q-TOF micro instrument. NMR spectra
were acquired in CD3OD on a Bruker Avance DRX-600 instrument
using standard pulse sequences and parameters. TLC was per-
formed on pre-coated silica-gel 60 A: Alugram UV254 Macherey-
Nagel in normal phase, and Silicagel 60 RP-18 F254S Merck in in-
versed phase. CC and vacuum liquid chromatography (VLC) were
realized on Kieselgel 60 (63-200 mesh), Merck. Flash chromatog-
raphy (FC) was carried out on a Grace Reveleris apparatus equipped
with a detector ELSD, a detector UV/vis and Reveleris Flash System
software. HPLC was performed on a Dionex apparatus equipped
▶Table 3 Minimal inhibitory concentration of extracts from Dolichandrone spathacea.
MIC of extract (mg/mL)
Microbial strain Petroleum
ether
CHCl3AcOEt MeOH Aqueous
MeOH
Vancomycine Gentamicine Amphop-
tericin B
Gram-positive bacteria
Bacillus subtilis ATCC 6633 51.2 510 10 S S NT
Enterococcus faecalis ATCC
1034
52.5 10 10 10 R R NT
Staphylococcus aureus CIP
8325-4
52.5 5NA 10 S S NT
Staphylococcus aureus CIP
53.154
52.5 52.5 10 S S NT
Micrococcus luteus (lab.
collection)
51.2 5 5 10 S S NT
Listeria innocua (lab.
collection)
52.5 5NA 10 S S NT
Staphylococcus epidermidis
(lab. collection)
≤ 0.3 1.2 2.5 10 10 S S NT
Streptococcus pyogenes
(lab. collection)
≤ 0.3 ≤ 0.3 ≤ 0.3 ≤ 0.3 ≤ 0.3 S S NT
Gram-negative bacteria
Providencia stuartii (lab.
collection)
51.2 5NA 10 R S NT
Pseudomonas aeruginosa
ATCC 9027
10 NA 10 10 10 R S NT
Shigella sonnei (lab.
collection)
≤ 0.3 ≤ 0.3 ≤ 0.3 ≤ 0.3 ≤ 0.3 I S NT
Proteus vulgaris (lab.
collection)
NA NA 10 NA 10 R S NT
Klebsiella pneumoniae (lab.
collection)
NA NA 10 NA NA R R NT
Serratia marcescens (lab.
collection)
NA NA 10 NA 10 R S NT
Escherichia coli CIP 54.127 NA NA 10 NA NA R S NT
Enterobacter cloacae (lab.
collection)
NA NA 10 NA NA R S NT
Salmonella enterica (lab.
collection)
NA NA 10 NA 10 R S NT
Yeas t
Candida glabrata (lab.
collection)
2.5 1.2 5NA 10 R R S
Candida tropicalis (lab.
collection)
510 5NA 10 R R S
Candida kefyr (lab.
collection)
510 10 NA NA R R S
Cryptococcus neoformans
(lab. collection)
10 NA 10 NA NA R R S
Candida albicans ATCC
2091
510 5NA 10 R R S
NA = not active; S = sensitive; I = intermediate sensitivity; R = resistant.
MIC (µg/mL) of positive controls: Gentamicin, S: ≤ 4, R: > 8; Vancomycin, S: ≤ 4, R: > 16; Amphotericin B, S: ≤ 1, R: > 4.
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Nguyen P-D et al. Antimicrobial Constituents from Leaves … Planta Med Int Open 2018; 5: e14–e23
Original Papers Thieme
with a LPG 3400AB pump, an ASI-100 auto-sampler, a diode array
detector UVD 340 S, an oven STH 585, and a Chromeleon software.
Phenomenex Luna C18 columns (5μ, 100 Å; 250 × 10 mm: column
1; 250 × 15 mm: column 2) and Interchrom Uptisphere Strategy
C18 column (2–5 μ, 250 × 10 mm: column 3) were used for semi-
preparative HPLC with gradient eluent [solvent A, H2O or H2O (pH
2.4, 0.025 % TFA); solvent B, MeCN or MeOH], and the chromato-
gram was monitored at 205 and 254 nm. Preparative HPLC chain
comprises a Merck column of 200 × 50 mm packed with C-18 silica
gel, an Armen AP 250/500 pump, an ACC 250/500 injector, and an
UV Merck K-2501 Knauer detector; fractions were collected using
a Büchi C-660 collector. Sugars were puried on a Waters chroma-
tographic chain with a Rezex ROA column (250 × 21.2 mm: column
4), Empower software, a 600 E pump, a 717 plus auto-sampler, and
a refractive index (RI) detector (flow rate = 3.5 mL/min, pres-
sure = 1400 psi); Chiralpak IC column (5 µm, 250 × 4.6mm: column
5) was used for the chiral identication of sugars (ow rate = 0.5 mL/
min, pressure = 300 psi).
Plant material
The leaves of D. spathacea were collected in Vinh Long Province, Vi-
etnam in December 2011. The plant was identied by Professor
Nghia Thin Nguyen (Faculty of Biology, University of Sciences
Hanoi, Vietnam) and a voucher specimen (CTU-CD001) was depos-
ited at the Department of Biology, Faculty of Pedagogy, Can Tho
University, Vietnam.
Extraction and isolation
Dried and powdered leaves (150 g) were macerated for 7 h with pe-
troleum ether (1.5 L) to furnish 0.98 g of extract (yield 0.65 %), and
treated in the same way successively with 1.5 L CHCl3 for 20 h (5 g;
yield 3.4 %), 1.5 L EtOAc (2 g; yield 1.4 %), 1.5 L MeOH (23 g; yield
15.3 %), and 1.5 L of 80 % aqueous MeOH (17 g; yield 12 %).
The MeOH extract (2 × 10 g) was subjected to silica gel VLC
(7 × 9.5 cm) using a gradient of solvent CHCl3 – MeOH – H2O
(100:0:0 – 0:100:5) to give 20 fractions. The p-methoxycinnamic
acid (480 mg) was obtained in VLC-fraction 4 eluted with CHCl3 –
MeOH (90:10).
VLC-fraction 5 [620 mg, eluted with CHCl3 – MeOH (85:15)] was
separated by C-18 FC using a gradient of MeOH – H2O (10:90 –
100:0); sub-fraction 5.9 [53 mg, eluted with MeOH – H2O (45:55)]
was puried by semi-preparative HPLC (column 2; 6 mL/min) using
an isocratic solvent of 25 % MeCN (pH = 2.4) during 30 min to yield
(2E,6E)-8-hydroxy-2,6-dimethyl-2,6-octadienoic acid (6.2 mg;
Rt = 10.66 min) and (2E,6Z)-8-hydroxy-2,6-dimethyl-2,6-octadie-
noic acid (3.5 mg; Rt = 11.31 min); sub-fraction 5.11 (40 mg) was
puried by the same way using an isocratic solvent of 28 % MeCN
to furnish a mixture of 6 R- and 6 S-(2E)-8-hydroxy-2,6-dimethyl-
2-octenoic acids (5.8 mg; Rt = 14.62 min).
VLC-fraction 6 [2.3 g, eluted with CHCl3 – MeOH (80:20)] was
chromatographed by silica gel FC using a gradient of CHCl3 – MeOH
(100:0 – 0:100) to obtain p-hydroxycinnamic acid (22 mg) and
6-(E)-p-methoxycinnamoyl-catalpol (240 mg); sub-fraction 6.3
[31 mg, eluted with CHCl3 – MeOH (93:7)] was puried by semi-
preparative HPLC (column 1; 3 mL/min) using an isocratic mode of
23 % MeCN (pH = 2.4) to give vanillic acid (1 mg; Rt = 7.13 min) and
isoferulic acid (2.3 mg; Rt = 12.39 min); the purication of sub-frac-
tion 6.5 [28 mg, eluted with CHCl3 – MeOH (91:9)] by semi-prepar-
ative HPLC (column 2; 6 mL/min) using a gradient solvent of 20-45 %
MeCN (pH = 2.4) gave p-hydroxybenzoic acid (3 mg; Rt = 8.69 min)
and luteolin (4.5 mg; Rt = 16.18 min); sub-fraction 6.7 (470 mg)
eluted with CHCl3 – MeOH (90:10) was separated by C-18 FC using
a gradient solvent of MeOH – H2O (10:90 – 100:0), then fractions
eluted with MeOH – H2O (60:40) were subjected to silica gel CC
using a gradient system of CHCl3 – MeOH (100:0 – 0:100) to ob-
tain 6-O-(p-methoxy-E-cinnamoyl)-ajugol (1) (50 mg) eluted with
5 % MeOH and a mixture of (E)- and (Z)-6-p-methoxycinnamoyl-
catalpol (2 mg; Rt = 25.46 min), nally puried by semi-preparative
HPLC (column 1, 4 mL/min) using an isocratic mode of 28 % MeCN.
VLC-fraction 7 [2.6 g, eluted with CHCl3 – MeOH (75:25)] was
chromatographed by preparative HPLC using a gradient of MeOH
– H2O (25:75 – 100:0); sub-fraction 7.5 [32 mg, eluted with MeOH
– H2O (35:65)] was separated by semi-preparative HPLC (column
3, 4 mL/min) using an isocratic of 28 % MeCN (pH = 2.4) to obtain a
mixture (Rt = 10.43 min) which was puried by a semi-preparative
HPLC using an isocratic of 17 % MeOH during 55 min to yield 6 S,9 S-
roseoside (6 mg; Rt = 46.98 min); the purication of sub-fraction 7.9
[90 mg, eluted with MeOH – H2O (35:65)] by semi-preparative HPLC
(column 3; 4 mL/min) using an isocratic mode of 23 % MeCN gave
minecoside (5 mg; Rt = 14.49 min) and 6-O-E-isoferuloyl-ajugol
(4 mg; Rt = 16.15 min); in the same way, sub-fraction 7.10 [87 mg,
eluted with MeOH – H2O (35:65)] yielded nemorososide (4 mg;
▶Table 4 Bioautography and Minimal Inhibitory Concentration of antimicrobial compounds of Dolichandrone spathacea.
Bioautography MIC (µg/mL)
Compound S. aureus
CIP 53.154
S. aureus
CIP 53.154
E. faecalis
ATCC 1034
S. pyogenes
(lab. collection)
P. aeruginosa
ATCC 9027
S. sonnei
(lab. collection)
decaeoylacteoside + + + 62.5 31.2 62.5 250 31.2
verbascoside + + + 125 31.2 125 250 31.2
luteolin + + 250 250 125 500 125
p-methoxycinnamic acid + + 62.5 125 62.5 125 62.5
minecoside NA NA 500 250 500 250
6-O-E-caeoylajugol + + 125 250 125 500 125
Vancomycin + + + 3.9 62.5 ≤ 1.9 62.5 7.8
Gentamicin + + + ≤ 1.9 15.6 1.9 7.8 ≤ 1.9
( + ): Anti-staphylococcal eect, rated from + (0.5 cm inhibition zone) to + + + + ( > 1.5 cm inhibition zone); NA: no activity.
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Nguyen P-D et al. Antimicrobial Constituents from Leaves … Planta Med Int Open 2018; 5: e14–e23
Rt = 14.70 min) and 6’’(Z)-nemoroside (7.5 mg; Rt = 19.02 min); the
6-O-p-E-coumaroyl-ajugol (23 mg; Rt = 25.19 min) was obtained
from sub-fraction 7.12 [50 mg, eluted with MeOH – H2O (40:60)]
by a semi-preparative HPLC (column 1; 4 mL/min) using an isocrat-
ic of 20 % MeCN; semi-preparative HPLC (column 3; 3.5 mL/min) of
sub-fraction 7.15 [62 mg, eluted with MeOH – H2O (60:40)] using
an isocratic of 25 % MeCN furnished mixture of 6’’R-(2E) -8-hy-
droxy-2,6-dimethyl-2-octenoyl)-ajugol (2) (7.5 mg; Rt = 22.54 min)
and mixture of 6’’R-(2E)-8-hydroxy-2,6-dimethyl-2-octenoyl)-ca-
talpol (3; 10 mg; Rt = 25.91 min); in the same way, sub-fraction 7.17
(52 mg, eluted with MeOH – H2O (65:35)) gave 6-O-(E) -cinnamoyl-
catalpol (14 mg; Rt = 20.72 min); the 28-O-β-D-glucopyranosyl un-
caric acid (4; 7.3 mg; Rf = 0.28) was obtained from sub-fraction 7.21
(27 mg, eluted with MeOH – H2O (100:0)) by using C-18 prepara-
tive TLC eluted with MeOH – H2O (70:30).
VLC-fraction 8 [2.2 g, eluted with CHCl3 – MeOH (70:30)] was
separated by preparative HPLC using a gradient mode of MeOH –
H2O (25:75 – 100:0) to yield verbascoside [397 mg, eluted with
MeOH – H2O (25:75)]; the purication of sub-fraction 8.4 [397 mg,
eluted with MeOH – H2O (35:65)] by semi-preparative HPLC (col-
umn 3; 4 mL/min) using an isocratic of 10 % MeCN gave decaeoyl-
acteoside (8 mg; Rt = 8.61 min); semi-preparative HPLC (column 1;
4 mL/min) applied to sub-fraction 8.12 [33 mg, eluted with MeOH
– H2O (40:60)] was carried out by using an isocratic mode of 20 %
MeCN to give verminoside (7 mg; Rt = 11.33 min); semi-preparative
HPLC (column 3, 3.5 mL/min) of sub-fraction 8.14 [40 mg, eluted
with MeOH – H2O (45:55)] yield isoverbascoside (14 mg;
Rt = 18.41 min) by using an isocratic mode of 19 % MeCN; sub-frac-
tion 8.15 [12 mg, eluted with MeOH – H2O (45:55)] was puried by
semi-preparative HPLC (column 3; 4 mL/min) using an isocratic
mode of 18 % MeCN to furnish luteolin-7-O-β-D-glucopyranoside
(7.5 mg; Rt = 15.81 min); 6-O-E-caeoyl-ajugol (7 mg; Rt = 24 min)
was obtained from sub-fraction 8.16 [40 mg, eluted with MeOH –
H2O (50:50)] by semi-preparative HPLC (column 1; 4 mL/min) using
an isocratic mode of 18 % MeCN; the same protocol applied to sub-
fraction 8.18 [33 mg, eluted with MeOH – H2O (55:45)] gave spe-
cioside (9 mg; Rt = 12.87 min) using an isocratic mode of 23 %MeCN;
the purication of sub-fraction 8.21 [90 mg, eluted with MeOH –
H2O (65:35)] yielded arjunglucoside I (6 mg; Rt = 13.13 min) and
nemoroside (9.5 mg; Rt = 17.16 min) by semi-preparative HPLC (col-
umn 3; 4 mL/min) using an isocratic mode of 25 % MeCN; sub-frac-
tion 8.23 [20 mg, eluted with MeOH – H2O (65:35)] was puried by
silica gel preparative TLC eluted with CHCl3 – MeOH – H2O (80:20/2)
to yield 28-O-β-D-glucopyranosyl-23-hydroxy-uncaric acid (5;
7.5 mg; Rf = 0.33).
VLC-fraction 13 [617 mg, eluted with CHCl3 – MeOH – H2O
(70:30:5)] was separated by C-18 FC using a gradient of MeOH –
H2O (10:90 – 100:0) to give 17 sub-fractions; luteolin-7-O-rutino-
side (6 mg; Rt = 14.85 min) was obtained by semi-preparative HPLC
(column 1; 3.5 mL/min) of sub-fraction 13.9 [18 mg, eluted with
MeOH – H2O (45:55)] using an isocratic solvent of 18 % MeCN
(pH = 2.4).
Purification of VLC-fraction 17 [90 mg, eluted with CHCl3 –
MeOH – H2O (30:70:5)] by semi-preparative HPLC (column 1; 4 mL/
min) using a gradient mode of 10 – 35 % MeCN (pH = 2.4) furnished
ixoside (6 mg; Rt = 5.79 min) and luteolin-7-O-glucuronide (4.5 mg;
Rt = 15.89 min).
The purities of all isolated compounds were ≥ 95 %, as deter-
mined by HPLC and 1H NMR.
Compound 1: amorphous powder; [α]D -143 (c 0.39, MeOH);
UV (MeOH) λmax nm (log ε) 202 (2.88), 226 (2.82), 308 (3.09); IR
(KBr) νmax cm − 1: 3428, 2931, 1706, 1662, 1632, 1575, 1514, 1170,
1002, 825; 1H- and 13C-NMR: see ▶Table 1; HR-ESI-MS m/z
531.1848 [M + Na] + (calcd. for C25H32O11Na 531.1842), 483.2442
[M + Na-MeO-OH] + , 185.2039 [Ajugol-Glc] + .
Compound 2: amorphous powder; [α]D -94 (c 0.25, MeOH); UV
(MeOH) λmax nm (log ε) 224 (3.34), 308 (2.33); 1H- and 13C-NMR:
see ▶Table 1; HR-ESI-MS m/z 539.2462 [M + Na] + (calcd. for
C25H40O11Na 539.2468).
Compound 3: amorphous powder; [α]D -107 (c 0.22, MeOH);
UV (MeOH) λmax nm (log ε) 224 (3.33), 308 (2.33); 1H- and 13C-
NMR: see ▶Table 1; HR-ESI-MS m/z 553.2256 [M + Na] + (calcd. for
C25H38O12Na 553.2261), 385.1032 [M + Na-Ester] + , 351.2457 [M-
Glc] + .
Compound 4: white powder; [α]D -18 (c 0.12, MeOH); UV
(MeOH) λmax nm (log ε) 210 (3.37), 308 (3.05); IR (KBr) νmax cm − 1:
3423, 2929, 1734, 1653, 1458, 1383, 1074; 1H- and 13C-NMR: see
▶Table 2; HR-ESI-MS m/z 673.3934 [M + Na] + (calcd. for C36H58O
10Na 673.3928), 457.2697 [M-Glc-OH] + .
Compound 5: white powder; [α]D -7 (c 0.19, MeOH); UV (MeOH)
λmax nm (log ε) 214 (3.43), 298 (2.83); IR (KBr) νmax cm − 1: 3422,
2927, 1732, 1598, 1455, 1384, 1072; 1H- and 13C-NMR: see ▶Table
2; HR-ESI-MS m/z 689.3869 [M + Na] + (calcd. for C36H58O11Na
689.3877), 503.3242 [M-Glc] + .
Acid hydrolysis
The MeOH extract (1 g) was reuxed in 25 mL of 2 N HCl in 4 h. After
extraction with EtOAc (3 × 25 mL), the aqueous layer was neutral-
ized with 0.5 M NaOH and freeze-dried. The glucose was identied
by comparison with an authentic sample by TLC using MeCOEt – i-
PrOH – MeCOMe – H2O (20/10/7/6). It was puried by semi-pre-
parative HPLC (column 4) using 2.5 μM H2SO4 as solvent, and iden-
tied by chiral analytic HPLC (column 5) using an isocratic mixture
of n-hexane/EtOH/TFA (80/20/1) in comparison with the authentic
samples of D-glucose (Rt = 19.44 min) and L-glucose
(Rt = 18.73 min).
Alkaline hydrolysis
4 mg of (2 or 3) were dissolved in 1 mL of 2 N NaOH and stirred over-
night. The solution was treated with Et2O (3 × 1 mL) to remove neu-
tral material. Acidication with dilute HCI was followed by extrac-
tion with Et2O (3 × 1 mL). The Et2O extract were evaporated in vacuo
and residue was puried by semi-preparative HPLC to give 1.2 mg
(or 1.5 mg) of monoterpenic acid.
Antimicrobial assays
After an overnight culture at 37 °C in Mueller-Hinton (MH) medi-
um, the bacteria (ATCC or CIP collections or clinical strains) and
yeasts (clinical strains) were resuspended in fresh MH medium up
to the working solution of 105 microorganisms/mL. The clinical
strains were part of the internal laboratory of microbiology or par-
asitology collections. All the antibiotics were used in serial dilutions
with the higher working concentration of 64 mg/L.
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Nguyen P-D et al. Antimicrobial Constituents from Leaves … Planta Med Int Open 2018; 5: e14–e23
Original Papers Thieme
To determine the minimum inhibitory concentration (MIC) of
the extracts, agar plates containing six dierent concentrations
(10, 5, 2.5, 1.2, 0.6, and 0.3 mg/mL) of each extract were loaded
with the different microorganism using a multiple inoculator
(Steers). After 24 h of incubation at 37 °C, the activity was estimat-
ed looking at the presence or absence of colonies. Solvents used to
prepare extracts and MH agar medium were checked for absence
of antibacterial activity (negative controls). Positive active controls
were gentamicin ( ≥ 99 % of purity) and vancomycin ( ≥ 99 % of pu-
rity) for antibacterial assays, and amphotericin B ( ≥ 99 % of purity)
for anti-yeast assays.
The immersion bioautography method was used to identify the
active compounds against S. aureus CIP 53.154. 10 μL of each com-
pound (2 mg/mL of methanol) or gentamicin (50 μg/mL, positive
control) were spotted onto TLC plates (10 × 10 cm). The plates were
than dried, sterilized and covered by the MH agar medium contain-
ing S. aureus (105 bacteria/mL) in square Petri dishes. After incuba-
tion for 24 h at 37 °C, bacterial growth was revealed by a 2 mg/mL
solution of thiazolylbluetetrazolium bromide (MTT) and growth in-
hibition zones were measured [39].
The broth microdilution method in 96-well plates was used to
determine MIC of active compounds against ve bacteria sensitive
to the extracts. Nine concentrations of each compound, from
500 μg/mL to 1.9 μg/mL in a serially twofold dilution, were tested
against 0.5 × 105 bacteria/mL, in a total volume of 200 μL, and in-
cubated overnight at 37 °C. Positive control wells with bacteria in
MH medium, gentamicin or vancomycin, as well as negative con-
trols such as medium alone or medium with methanol were sys-
tematically added to the test. Bacterial growth was followed visu-
ally and by spraying MTT incubating at 37 °C for at least 10 min. The
MIC value was determined as the lowest concentration of a com-
pound leading to a clear well. This test was performed in triplicate.
Supporting information
Tables with 1H- and 13C-NMR data of the 30 known compounds,
and 1H- and 13C-NMR Spectra of compounds (1-5) are available as
Supporting Information.
Acknowledgments
The authors wish to thank Dominique Harakat (ICMR) for record-
ing mass spectra, Prof. Nghia Thin Nguyen for the plant identica-
tion, Dien Trung Nguyen and Hoang Viet Ho (Can Tho University)
for the plant collection, Dr. Georges Massiot (ICMR) for stimulating
discussions, Prof. Christophe De Champs and Janick Madoux (Lab-
oratory of Bacteriology, CHU Reims) for access to multiple inocu-
lators material, and Prof. Jerome Depaquit (Laboratory of parasi-
tology-mycology, University of Reims) for providing yeasts.
Conflict of Interest
The authors declare no conict of interest.
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