Diterpenes and phenylpropanoids from clerodendrum splendens

Article abstract of DOI:10.1055/s-0033-1350648

Four new clerodane diterpenes (1-4) and one new phenylpropanoid (5) have been isolated from Clerodendrum splendens aerial parts, together with nine known compounds. Their structures were determined by spectroscopic and spectrometric analysis and chemical

Full text of DOI:10.1055/s-0033-1350648

Original Papers 1341
Diterpenes and Phenylpropanoids from
Clerodendrum splendens
Authors
Laura Faiella1, Abeer Temraz2, Roberta Cotugno3, Nunziatina De Tommasi3, Alessandra Braca1
1
Affiliations
Dipartimento di Farmacia, Università di Pisa, Pisa, Italy
Faculty of Pharmacy, Al Azhar University, Nasr-City, Cairo, Egypt
Dipartimento di Farmacia, Università di Salerno, Salerno, Italy
2
3
Key words
Abstract
was determined by Snatzkeʼs method. Antiprolif-
erative activity of diterpenes in HeLa cells was al-
so evaluated. The IC50 values were 98 11 µM for
"
l Clerodendrum splendens
!
"
l Verbenaceae
Four new clerodane diterpenes (1–4) and one
new phenylpropanoid (5) have been isolated from
Clerodendrum splendens aerial parts, together
with nine known compounds. Their structures
were determined by spectroscopic and spectro-
metric analysis and chemical methods. The abso-
lute configuration of the 15,16-diol moiety in 4
"
l clerodane diterpenes
3 and 101 8 µM for 1, respectively.
"
l antiproliferative activity
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
Introduction
roots and leaves are used for the treatment of skin
diseases, cancer, syphilis, gonorrhea, ulcers, and
various inflammatory diseases [4]. Previous stud-
ies on this species reported the presence of flavo-
noids and neo-clerodane diterpenoids [4–7]. As a
part of a program to search for bioactive diter-
penes, the aerial parts of C. splendens were inves-
tigated. Four new clerodane diterpenes, 2α-ace-
toxy-3β-(2′,3′-diacetoxy-2′-methyl)-butanoy-
loxy-14-hydro-15-hydroxyclerodin (1), 3β,15-di-
!
The genus Clerodendrum L., Verbenaceae family, is
very widely distributed in tropical and subtropi-
cal regions of the world and comprises about 580
species of small trees, shrubs, and herbs [1]. The
Latin name used in the beginning was “Cleroden-
drum”, then giving place to the Greek form “Cler-
odendron”; in Greek “Klero” means chance and
“Dendron” means tree. Later the Latinized name
“Clerodendrum” was readopted and is now com-
monly used by taxonomists for the classification
and description of the genus and species [2].
Many indigenous systems of medicines used vari-
ous species of this genus as anti-inflammatory,
antidiabetic, antimalarial, antiviral, antihyperten-
sive, and hypolipidemic remedies. The tribal use
of C. inerme as an antidote of poisoning from fish,
crabs, and toads was also known [3]. In the last
decade, this genus has attracted the attention of
phytochemists due to the many monoterpenes,
sesquiterpenes, diterpenes, triterpenes, and iri-
doids that have been isolated. Particularly, the
Clerodendrum genus is one of the major sources
of neo-clerodane diterpenoids, a large group of
bioactive natural compounds isolated mainly
from Asteraceae, Lamiaceae, and Verbenaceae [3].
Clerodendrum splendens G. Don is a climbing ever-
green brush plant with attractive red flowers na-
tive to Sierra Leone (West Africa) and is used in
traditional Nigerian medicine. Decoctions of the
hydroxy-14-hydro-clerodin
(2), 2α,15-dihy-
droxy-3β-(2′-hydroxy-2′-methyl-3′-acetoxy)-bu-
tanoyloxy-6α,18-diacetoxy-4α,17-epoxy-clero-
dan-11,16-lactone (3), and 3β,14S,15-trihydroxy-
6α,18-diacetoxy-4α,17-epoxy-clerodan-11,16-
lactone (4), as well as one new phenylpropanoid
β-(3,4-dihydroxyphenyl)ethyl-O-β-D-xylopyran-
osyl-(1 → 2)-α-L-rhamnopyranosyl-(1 → 3)-6-O-t-
received
revised
accepted
May 5, 2013
June 12, 2013
June 25, 2013
Bibliography
DOI http://dx.doi.org/
10.1055/s-0033-1350648
Published online August 8, 2013
Planta Med 2013; 79:
1341–1347 © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
"
caffeoyl-β-D-glucopyranoside (5) (l Fig. 1), along
with nine known compounds, 14,15-dihydro-15-
hydroxy-3-epicarioptin [8], phlinoside B [9], ver-
bascoside [10], isoacteoside [11], hispidulin-7-O-
β-D-glucopyranoside [12], hispidulin-7-O-neo-
hesperoside [13], luteolin-7-O-neohesperoside
[14], rosmarinic acid [15], and 2β-angeloyloxy-
5β-hydroxy-7α,10β-methyl-eudesm-3-ene-1-
one [16], were isolated. The antiproliferative ac-
tivity of diterpenes against HeLa tumor cells was
also investigated.
Correspondence
Prof. Nunziatina De Tommasi
Dipartimento di Farmacia
Università di Salerno
Via Ponte Don Melillo
84084 Fisciano (Salerno)
Italy
Phone: + 39089969754
Fax: + 39089969602
detommasi@unisa.it
Faiella L et al. Diterpenes and Phenylpropanoids… Planta Med 2013; 79: 1341–1347

1342 Original Papers
Fig. 1 Structures of compounds 1–5.
Results and Discussion
acetoxy-2-methylbutanoyloxy moiety were attached at C-2 and
C-3, respectively. Elucidation of the relative stereochemistry of 1
was mostly based on the close similarities of its NMR data to
those of similar compounds [8,19]. Thus, compound 1 was as-
signed as a mixture of C-15 epimers of 2α-acetoxy-3β-(2′,3′-diac-
etoxy-2′-methyl)-butanoyloxy-14-hydro-15-hydroxyclerodin.
The molecular formula of compound 2 (C24H36O9) was estab-
lished by 13C NMR and HR ESIMS spectrum (m/z 469.2500 for
[M + H]+). In the ESI MS spectrum, two main fragments at m/z
425 [M – H – 42]− and 383 [M – H – 42–42]− due to the loss of
two C2H2O moieties were also observed. Its NMR spectral data
!
Compound 1’s molecular formula was determined as C35H50O16
from its HR ESIMS (m/z 727.3204 [M + H]+) and 13C NMR data.
The ESI MS spectrum of 1 showed a quasimolecular ion peak at
m/z 749 [M + Na]+ and peaks at m/z 689 [M + Na – 60]+, 531 [M +
Na – 218]+, 471 [M + Na – 218–60]+, and 411 [M + Na – 218–60–
60]+ due to the loss of two acetyl groups and an esterified acyl
group. The 1H and 13C NMR spectra (l Table 1) revealed the pres-
"
ence of five acetyl groups, a methine doublet (δ 1.20, d, J = 6.5 Hz)
coupled with a methyne quartet (δ 5.10, q, J = 6.5 Hz), and a meth-
yl singlet (δ 1.58) arisen from a 2,3-diacetoxy-2-methylbutanoy-
loxy moiety [17], together with the characteristic signals of a
neo-clerodane diterpene having a 4α,17-oxirane bicycle (δH 2.63,
2.91 [H2-17], δC 41.8 [C-17]; δC 64.0 [C-4]). Analysis of 2D NMR
spectra showed that compound 1 possessed a hexahydrofurofu-
ran ring moiety typical of 14-hydroclerodin [18]. In fact, protons
H-11, H-15, and H-16 appeared as a pair of signals: H-11 δ 4.04
and 4.65 (0.5 H each); H-15 δ 5.33 and 5.45 (0.5 H each), and H-
16 δ 5.61 and 5.65 (0.5 H each). The 13C NMR spectrum of 1 was in
complete agreement with the proposed structure and with the
previously reported 13C NMR data of other neo-clerodane diter-
penes isolated from the genus Clerodendrum containing the same
C-11/C-16 moiety [8,18]. Indeed, with the help of 1D TOCSY and
DQF COSY spectra, it was possible to attribute the values of pro-
tons and carbons of both epimers. The elucidation of the whole
skeleton was achieved on the basis of 1D TOCSY, DQF COSY,
HSQC, and HMBC correlations, which also allowed the assign-
ment of all the resonances in the 13C NMR spectrum of the perti-
nent carbons. HMBC correlations from H-1 to C-2, C-5 and C-10,
from H-3 to C-4 and C-17, from H-16 to C-11, C-13, and C-15,
from H-12 to C-13, C-14, and C-16, and from H-18 to C-4, C-5, C-
6, and C-10, confirmed the assignment of the neo-clerodane ring
carbons. The peak correlating signals at δ 5.46 (H-3) and
169.2 ppm, δ 1.58 (Me-5′) and 169.2 and 83.5 ppm, and δ 4.90
(H-2) and 172.1 ppm showed that an acetoxy group and a 2,3-di-
"
(l Table 1) suggested that the structure of 2 resembled that of 1
but differed in the A ring substitutions. The observed differences
in the NMR spectra of 2 and 1 were consistent with the presence
in 2 of a hydroxyl group at C-3 and no other substituents on ring
A. Thus, the structure of 2 was deduced to be a mixture of C-15
epimers of 3β,15-dihydroxy-14-hydro-clerodin.
Compound 3 (C31H46O14) showed a quasimolecular ion peak at
m/z 643.2899 [M + H]+ in the positive HR ESIMS. The prominent
fragment ion observed in the ESI MS spectrum at m/z 486 [M + Na
– 176]+ suggested the presence of a 2′-hydroxy-3′-acetoxy-2′-
methylbutanoyl moiety in 3. Analysis of the 1H and 13C NMR spec-
"
tra (l Table 1) of 3 clearly indicated that it also belonged to the
1
neo-clerodane diterpenoid class. The H NMR spectrum showed
signals due to one tertiary methyl (δ 1.02, 3H, s), one secondary
methyl (δ 0.93, 3H, J = 6.0 Hz), three acetate residues (δ 1.91.
1.99, and 2.11, each 3H, s), a methyl doublet (δ 1.27, d, J = 6.5 Hz)
coupled with a methine quartet (δ 5.12, q, J = 6.5 Hz), and a meth-
yl singlet (δ 1.29, s) arising from the 2′-hydroxy-3′-acetoxy-2′-
methylbutanoyloxy moiety at the C-3β position. It also showed
the two AB quartets, typical of a primary carbinol methylene
group at δ 4.51 and 4.65 (1H, J = 12.0 Hz, H-18a and H-18b), an
epoxide methylene group at δ 2.64 and 2.90 (1H, J = 4.0 Hz, H-
17a and H-17b), a doublet of doublets at δ 4.82 (1H, J = 11.0,
4.5 Hz, H-6) due to a C-6 proton, a multiplet at δ 3.64 (1H, m, H-
2), and a doublet at δ 5.15 (1H, J = 10.0 Hz, H-3) due to a C-3 pro-
Faiella L et al. Diterpenes and Phenylpropanoids… Planta Med 2013; 79: 1341–1347

Original Papers 1343
Table 1 1H and 13C NMR data of compounds 1–4 (CD3OD, 600 MHz, J in Hz).
Position
1
2
3
4
δH
δC
28.0
δH
δC
21.1
δH
δC
32.0
δH
δC
1a
1b
2a
2b
3
2.75 m
2.28 m
2.19 m
1.85 m
22.7
1.85 m
4.90a
–
1.75 m
–
1.83 m
–
–
–
71.6
–
1.82 m
31.3
–
3.64 m
71.0
–
2.12 br dd (11.5, 4.5)
34.3
–
–
1.30 m
–
1.40 m
5.46 d (10.5)
–
71.2
64.0
47.0
72.0
32.4
–
3.98 dd (11.5, 5.0)
–
65.4
68.0
49.4
71.7
33.0
–
5.15 d (10.0)
74.4
64.0
47.2
72.2
33.7
–
4.02 dd (11.5, 5.5)
66.3
68.4
47.9
73.0
34.3
–
4
–
–
5
–
–
–
–
6
4.87 dd (11.0, 4.5)
1.92 m
4.80 dd (11.0, 4.5)
1.70 m
4.82 dd (11.0, 4.5)
1.70 m
4.84 dd (11.0, 5.0)
1.75 m
7a
7b
8
1.60 m
1.42 m
1.50 m
1.50 m
1.55 m
37.0
41.6
43.0
84.0
85.1
33.7
–
1.52 m
35.8
42.0
47.6
83.6
83.7
32.8
–
1.67 m
35.6
42.5
43.1
89.9
1.65 m
35.5
42.8
48.0
88.5
9
–
–
–
–
10
11
1.92 dd (10.0, 3.0)
4.65 dd (11.0, 6.2)
4.04 dd (12.0, 5.0)
1.70 m
1.78 dd (10.0, 2.5)
4.63 dd (11.0, 6.2)
3.99 dd (12.0, 5.0)
2.06 m
1.95 dd (9.5, 3.0)
4.70 t (9.0)
1.92 dd (10.0, 3.9)
4.72 t (9.0)
12a
12b
13
2.37 br dd (13.0, 9.0)
2.06 m
28.4
–
2.31 br dd (13.0, 9.0)
23.0
–
1.48 m
1.61 m
2.22 m
3.11 m
41.0
40.6
–
2.97 m
40.2
39.7
–
2.86 m
39.9
34.3
–
2.90 m
45.0
72.6
–
14a
14b
15a
2.26 m
2.07 m
1.95 m
4.06 m
1.78 m
1.78 m
1.80 m
–
5.45 br d (5.5)
5.33 br d (3.5)
–
99.3
99.6
–
5.54 br d (4.6)
5.45 br d (5.0)
–
98.5
97.9
–
3.72 m
60.0
3.60 dd (12.0, 8.0)
64.9
15b
16
3.67 m
–
3.49 dd (12.0, 7.0)
–
5.65 d (5.5)
5.61 d (5.5)
2.91 d (4.0)
2.63 d (4.0)
4.82 d (12.3)
4.52 d (12.3)
0.94 s
109.5
108.2
41.8
–
5.71 d (4.7)
5.67 d (6.2)
2.84 d (3.5)
2.72 d (3.5)
4.91 d (12.5)
4.33 d (12.5)
0.95 s
106.8
108.7
41.6
–
–
181.0
–
181.0
17a
2.90 d (4.0)
2.64 d (4.0)
4.65 d (12.0)
4.51 d (12.0)
1.02 s
43.2
–
2.90 d (4.5)
2.75 d (4.5)
4.94 d (12.0)
4.36 d (12.0)
1.03 s
43.2
–
17b
18a
62.7
–
61.6
–
62.5
–
62.8
–
18b
19
15.0
15.2
21.3
21.3
20.3
171.8
172.1
170.0
169.2
83.5
73.0
15.0
15.2
21.3
21.2
171.9
170.5
13.0
15.9
19.7
20.2
13.8
16.2
21.3
21.2
14.0
16.4
21.2
21.4
20
0.96 d (6.0)
2.10 s
0.92 d (6.0)
2.10 s
0.93 d (6.0)
2.11 s
0.96 d (6.5)
2.12 s
OCOCH3
2.10 s
1.93 s
1.99 s
1.96 s
1.97 s
OCOCH3
–
–
172.8
171.2
–
172.6
171.5
–
172.7
171.4
1′
2′
–
–
–
–
–
–
–
–
172.0
77.0
75.0
13.6
22.0
21.2
–
–
–
–
–
–
–
–
3′
5.10 q (6.5)
1.20 d (6.5)
1.58 s
2.10 s
1.92 s
–
5.12 q (6.5)
1.27 d (6.5)
1.29 s
4′
5′
COCH3
1.91 s
COCH3
–
–
171.7
–
Data assignments were confirmed by DQF-COSY, 1D-TOCSY, HSQC, and HMBC experiments. a Overlapped signal
ton. 1D TOCSY and DQF COSY measurements showing couplings
between H-11, H2-12, H-13, H2-14, and H2-15 established the
spin system C-11–C-15, indicating the presence of a β-substitu-
ent carrying a five membered lactone ring. The linkage between
C-14 and the butanolide lactone ring was deduced by HMBC cor-
relations; the signal at δ 4.70 (1H, t, J = 9.0 Hz, H-11) correlated to
C-8, C-9, C-10, and C-13; δ 2.86 (1H, m, H-13) to C-12, C-15, and
C-16; δ 3.72 (1H, m, H-15a) to C-14 and C-15. Signals attributable
to the three acetate groups appeared at δ 171.5, 171.7, 172.6, and
at δ 21.2 (2 CH3) and 21.3, while the signals due to carbons bear-
ing the acetate groups appeared at δ 62.5 (C-18), 72.2 (C-6), and
75.0 (C-3′). The elucidation of the whole skeleton from the above
subunits was achieved on the basis of 1D TOCSY, DQF COSY,
HSQC, and HMBC correlations, which also allowed the assign-
ment of all the resonances in the 13C NMR spectrum. The relative
stereochemistry of rings A and B was established by comparison
of NMR data to those of similar compounds [8,19]; the relative
stereochemistry at C-11 and C-13 was indicated by a 2D ROESY
experiment showing ROE cross-peaks between Me-20 and H-
12a and H-14a and between H-11 and Me-19 and Me-20. Conse-
quently,
3 was characterized as 2α,15-dihydroxy-3β-(2′-hy-
droxy-2′-methyl-3′-acetoxy)-butanoyloxy-6α,18-diacetoxy-
4α,17-epoxy-clerodan-11,16-lactone.
Faiella L et al. Diterpenes and Phenylpropanoids… Planta Med 2013; 79: 1341–1347

1344 Original Papers
the 1,2-diol moiety. In particular an S-monosubstituted glycol
gives rise to a positive Cotton effect at 305 nm, while a negative
sign leads to the assignment of R-configuration. Thus, the
305 nm sign observed in the CD spectrum of 4 allowed us to as-
sign the S-configuration to C-14. Therefore, compound 4 was as-
signed the structure of 3β,14S,15-trihydroxy-6α,18-diacetoxy-
4α,17-epoxy-clerodan-11,16-lactone.
The absolute stereochemistry of compounds 1–4 is assumed,
while the side chain stereochemistry of 1 and 3 was not deter-
mined.
Table 2 1H and 13C‑NMR data of compound 5 (CD3OD, 600 MHz, J in Hz).
Position
5
δH
δC
Aglycone 1
–
130.5
115.9
146.2
145.0
116.8
121.2
36.3
–
2
3
4
5
6
α
6.64 d (2.0)
–
–
6.70 d (8.0)
6.56 dd (8.0, 2.0)
3.76 m
Compound 5’s molecular formula was determined as C34H44O19
by HR ESI mass spectrometry (m/z 757.2637 [M + H]+). Its ESI
MS spectrum showed a quasimolecular ion peak at m/z 779 [M +
Na]+. Two main fragments at m/z 647 [M + Na – 132]+ and 501 [M
+ Na – 132–146]+, due to the subsequent loss of one pentose and
one deoxyhexose moiety, were also observed. The 13C NMR spec-
3.96 m
β
2.78 m
72.1
104.1
74.8
83.8
70.5
75.0
64.4
–
Glc 1
4.36 d (7.8)
3.55 dd (9.0, 7.8)
3.50 t (9.0)
3.45 t (9.0)
3.28 m
2
3
4
5
"
trum (l Table 2) of 5 displayed 34 signals, of which 17 were as-
6a
4.52 dd (12.0, 3.0)
4.36 dd (12.0, 4.5)
5.45 d (1.8)
3.99 dd (3.0, 1.8)
3.77 dd (9.0, 3.0)
3.38 t (9.0)
3.98 m
signed to the aglycone moiety; the remaining 17 signals corre-
sponded to two hexoses and one pentose sugar residues. The 1H
6b
Rha 1
101.6
82.3
72.1
74.0
69.7
17.7
107.3
74.5
77.8
70.5
67.0
–
"
NMR spectrum (l Table 2) of 5 displayed proton signals charac-
2
teristic of a trans-caffeoyl group [three aromatic protons resonat-
ing at δ 6.80 (1H, d, J = 8.0 Hz), 6.90 (1H, dd, J = 8.0, 2.0 Hz), 7.05
(1H, d, J = 2.0 Hz) as an ABX system and two trans olefinic protons
as an AB system at δ 6.30, 7.58 (1H, d, J = 16.0 Hz)] and a 3,4-dihy-
droxyphenylethanol moiety [three aromatic protons at δ 6.56
(1H, dd, J = 8.0, 2.0 Hz), 6.64 (1H, d, J = 2.0 Hz), 6.70 (1H, d,
J = 8.0 Hz) as an ABX system and an A2B2 system assigned to a hy-
droxyethyl group at δ 2.78 (2H, m), 3.76 (1H, m), 3.96 (1H, m)]
[22]. Additionally, three signals assignable to anomeric protons
indicated the presence of three sugar moieties: a doublet at δ
4.36 (1H, J = 7.8 Hz, H-1 of glucose), a broad singlet at δ 5.45 (1H,
J = 1.8 Hz, H-1 of rhamnose), and one doublet at δ 4.34 (1H,
J = 7.5 Hz, H-1 of xylose), consistent, with the help of the 13C
NMR spectrum, with the following C-1 configuration: β for glu-
cose, α for rhamnose, and β for xylose. These findings matched
those in the HSQC/13C NMR spectra, where three corresponding
anomeric carbons resonated at δ 104.1, 101.6, and 107.3, respec-
tively. Other remaining 1H and 13C NMR signals were assigned
with the aid of 2D NMR spectra. The downfield shift of C-3′
(83.8 ppm) of glucose indicated that this position was a glycosy-
lation site. This finding was further confirmed by an HMBC ex-
periment, where a cross-peak between H-1rha (δ 5.45) and C-3glc
(83.8 ppm) was observed. The carbon resonances assigned to the
β-xylose unit suggested its terminal position. A further site of
connectivity was proved to be C-2 of rhamnose, on the basis of
1H and 13C NMR spectra, as well as HMBC correlations between
H-1xyl (δ 4.34) and C-2rha (82.3 ppm). The acylation site was on
C-6 of glucose as evidenced by the strong deshielding of H-6aglc
and H-6bglc at δ 4.52 and 4.36 and the cross-peak between H-
6aglc and H-6bglc and COO at 169.0 ppm. The configuration of
the sugar units was assigned after hydrolysis of 5 with 1 N HCl
and GC analysis of trimethylsilylated sugar through a chiral col-
umn. On the basis of this data, compound 5 was determined to
be the new phenylpropanoid glycoside β-(3,4-dihydroxyphenyl)
ethyl-O-β-D-xylopyranosyl-(1 → 2)-α-L-rhamnopyranosyl-
3
4
5
6
1.24 d (6.5)
4.34 d (7.5)
3.38 dd (9.0, 7.5)
3.90 t (9.0)
3.47 m
Xyl 1
2
3
4
5a
3.86 dd (12.0, 3.0)
3.22 dd (12.0, 4.5)
–
5b
Caffeic acid 1
127.8
115.0
149.7
145.6
116.4
123.2
114.7
147.5
169.0
2
7.05 d (2.0)
–
3
4
–
5
6.80 d (8.0)
6.90 dd (8.0, 2.0)
6.30 d (16.0)
7.58 d (16.0)
–
6
α
β
COO
Data assignments were confirmed by DQF-COSY, 1D-TOCSY, HSQC, and HMBC ex-
periments
The HR ESIMS of 4 showed a quasimolecular ion [M + H]+ at m/z
485.2423, consistent with a molecular formula of C24H36O10. In
the ESI MS spectrum, two fragments at m/z 447 [M + Na – 60]+
and 387 [M + Na – 60–60]+ indicated the loss of two acetoxy res-
"
idues. Its NMR spectral data (l Table 1) suggested that the struc-
ture of 4 resembled that of 3 but differed in the shifts of A ring
signals and shifts of H-13/C-13, H-14/C-14, H-15/C-15 (2.90/45.0
in 4 vs. 2.86/39.9 in 3, 4.06/72.6 in 4 vs. 1.95, 1.80/34.3 in 3, 3.49,
3.60/64.9 in 4 vs. 3.67, 3.72/60.0 in 3, respectively). In particular
1H and 13C NMR signals due to the A ring atoms were different.
The NMR spectra of 4 contained one less hydroxyl group and acyl
moiety signals. The 1D TOCSY and DQF COSY showed the pres-
ence of a hydroxyl group at C-3. Another difference was the pres-
ence in 4 of a 14,15-diol moiety. The absolute configuration of the
14,15-diol moiety of 4 was determined by the circular dichroism
(CD) induced after in situ complexation with dimolybdenum tet-
racetate in DMSO solution [20]. According to a rule proposed by
Snatzke [21], the sign of the diagnostic band at about 305 nm cor-
related with the absolute configuration of the chiral centers in
(1 → 3)-6-O-t-caffeoyl-β-D-glucopyranoside.
On the basis of literature evidence on related compounds [6], the
isolated clerodane diterpenes were tested for antiproliferative
activity, exposing HeLa cells to 50 µM of each chemical for 24 h
and 15 µM of phenethylisothiocyanate (PEITC) as control. Only
compounds 3 and 1 displayed any cell growth inhibition activity.
Further experiments were performed to study the activity of 1
Faiella L et al. Diterpenes and Phenylpropanoids… Planta Med 2013; 79: 1341–1347

Original Papers 1345
and 3 by using different doses (range 20–100 µM). The IC50 values
calculated from cell viability dose-response curves obtained by
incubating HeLa cells for 72 h were 98 11 µM for 3 and 101
8 µM for 1.
20 min), and hispidulin-7-O-neohesperoside (1 mg, tR = 38 min).
Fraction E (248 mg, 1350–1410 mL), fraction G (77 mg, 1545–
1620 mL), and fraction I (115 mg, 1725–1830 mL) were directly
subjected to RP-HPLC with MeOH–H2O (2:3) as the eluent to give
pure verbascoside (2.4 mg, tR = 13 min), compound 5 (5.6 mg,
tR = 20 min), hispidulin-7-O-neohesperoside (7.4 mg, tR = 38 min)
from fraction E, isoacteoside (5.1 mg, tR = 20 min) from fraction G,
and luteolin-7-O-neohesperoside (2.2 mg, tR = 29 min) and hispi-
dulin-7-O-β-D-glucopyranoside (2 mg, tR = 45 min) from fraction
I. Fraction N (120 mg, 1950–1995 mL) was purified by RP-HPLC
with MeOH–H2O (35:65) as the eluent to give pure rosmarinic
acid (6.6 mg, tR = 35 min). Part of the CHCl3–MeOH extract (10 g)
was chromatographed over Sephadex LH-20 column (5 × 70 cm,
flow rate 2.0 mL/min) using MeOH as the eluent and collecting
164 fractions of 20 mL that were grouped by TLC into twelve ma-
jor fractions (A–N). Fraction F (189 mg, 1220–1480 mL) was puri-
fied by RP-HPLC with MeOH–H2O (35:65) as the eluent to yield
phlinoside B (11.9 mg, tR = 22 min). Fraction H (70 mg, 2040–
2200 mL) was also purified by RP-HPLC with MeOH–H2O (2:3)
as the eluent to yield hispidulin-7-O-β-D-glucopyranoside
(8.6 mg, tR = 38 min). Part of the CHCl3 extract (10 g) was chroma-
tographed over silica gel column (9 × 20 cm) eluting with CHCl3
followed by increasing concentrations of MeOH in CHCl3 (be-
tween 1% and 50%, CHCl3 7.5 L, CHCl3-MeOH 99:1 3 L, CHCl3-
MeOH 98:2 4 L, CHCl3-MeOH 97:3 1.5 L, CHCl3-MeOH 95:5
3.5 L, CHCl3-MeOH 9:1 1 L, CHCl3-MeOH 1:1 1 L). Fractions of
75 mL were collected and grouped by TLC into 18 fractions (A–
T). Fraction H–L (1.6 g, 5.5–7.5 L) was purified by RP-HPLC with
MeOH–H2O (7:3) as the eluent to yield 2β-angeloyloxy-5β-hy-
droxy-7α,10β-methyl-eudesm-3-ene-1-one (1.5 mg, tR = 19 min).
Fraction O (500 mg, 12.4–13 L) was purified by RP-HPLC with
MeOH–H2O (3:2) as the eluent to yield 14,15-dihydro-15-hy-
Materials and Methods
!
General experimental procedures
Optical rotations were measured on a Perkin-Elmer 241 polarim-
eter equipped with a sodium lamp (589 nm) and a 1 dm micro-
cell. UV spectra were recorded on a Perkin-Elmer-Lambda spec-
trophotometer. CD spectra were measured on a JASCO J-810
spectropolarimeter with a 0.1-cm cell in DMSO at room temper-
ature under the following conditions: speed 50 nm/min, time
constant 1 s, bandwidth 2.0 nm. NMR experiments were per-
formed on a Bruker DRX-600 spectrometer at 300 K. 2D NMR
spectra were acquired in CD3OD in the phase-sensitive mode
with the transmitter set at the solvent resonance and TPPI (time
proportional phase increment) was used to achieve frequency
discrimination in the ω1 dimension. The standard pulse sequence
and phase cycling were used for DQF-COSY, TOCSY, HSQC, HMBC,
and NOESY experiments. The NMR data were processed on a Sili-
con Graphic Indigo2 Workstation using UXNMR software. ESIMS
were obtained using a Finnigan LC‑Q Advantage Termoquest
spectrometer, equipped with Xcalibur software. HRESIMS were
acquired in the positive ion mode on a Q‑TOF premier spectrom-
eter (Waters-Milford). TLC was performed on precoated Kieselgel
60 F254 plates (Merck); compounds were detected by spraying
with Ce(SO4)2/H2SO4 (Sigma Aldrich) and NTS (Naturstoffe re-
agent)-PEG (Poliethylene glycol 4000) solutions. Column chro-
matography was performed over Sephadex LH-20 (Pharmacia);
reversed-phase (RP) HPLC separations were conducted on a Shi-
madzu LC-8A series pumping system equipped with a Shimadzu
RID10A refractive index detector and a Shimadzu injector, using a
C18 µ-Bondapak column (30 cm × 7.8 mm) and a mobile phase
consisting of MeOH‑H2O mixtures at a flow rate of 2 mL/min. GC
analyses were performed using a Dani GC 1000 instrument. Di-
molybdenum tetracetate was purchased from Sigma Aldrich.
droxy-3-epicarioptin (5.5 mg, tR = 11 min) and compound
1
(5.4 mg, tR = 22 min). Fraction Q (292 mg, 17.3–17.8 L) was puri-
fied by RP-HPLC with MeOH–H2O (2:3) as the eluent to yield
compound 2 (1.8 mg, tR = 27 min). Fractions R (294 mg, 19.7–
20.2 L) and S (270 mg, 20.3–20.4 L) were purified by RP-HPLC
with MeOH–H2O (45:55) as the eluent to yield compound 3
(1.8 mg, tR = 20 min) from fraction R and compound 4 (1.3 mg,
tR = 12 min) from fraction S. All the compounds met the criteria
of ≥ 95% purity, as inferred by HPLC and NMR analyses.
Plant material
Aerial parts of C. splendens G. Don were collected in El Zoharia Re-
search Garden of Cairo, Egypt, in March 2010 and identified by
Dr. Mamdouh Shokry (El Zoharia Research Garden, Cairo, Egypt).
The voucher specimen (No. 7191 C. splendens G. Don/1) was de-
posited at the Herbarium Hortii Botanici Pisani, Flora Aegyptiaca,
Pisa, Italy.
2α-acetoxy-3β-(2′,3′-diacetoxy-2′-methyl)-butanoyloxy-14-hy-
dro-15-hydroxyclerodin (1): colorless amorphous powder; [α]D25
− 9.4 (c 0.45, MeOH); 1H and 13C NMR data, see l Table 1; ESI MS
"
m/z 749 [M + Na]+, 689 [M + Na – 60]+, 531 [M + Na – 218]+, 489
[M + Na – 218–42]+, 471 [M + Na – 218–60]+, 411 [M + Na – 218–
60–60]+; HR ESIMS [M + H]+ 727.3204 (calcd. for C35H51O16
727.3177).
Extraction and isolation
3β,15-dihydroxy-14-hydro-clerodin (2): colorless amorphous
powder; [α]2D5 + 5.7 (c 0.15, MeOH); 1H and 13C NMR data, see
l Table 1; ESI MS m/z 491 [M + Na] , 431 [M + Na – 60] , 371 [M
+ Na – 60–60]+, 467 [M – H]−, 425 [M – H – 42]−, 383 [M – H – 42–
42]−; HR ESIMS m/z 469.2500 [M + H]+ (calcd. for C24H37O9
469.2438).
Dried powdered aerial parts of C. splendens (775 g) were succes-
sively and separately extracted for 48 h with n-hexane, CHCl3,
CHCl3–MeOH (9:1), and MeOH, by exhaustive maceration (2 L),
to give 15.6, 13.5, 21.7, 26.8 g of the respective residues. The
MeOH extract was partitioned between n-BuOH and H2O, to af-
ford an n-BuOH residue (5.7 g). The n-BuOH fraction was submit-
ted to a Sephadex LH-20 column (5 × 70 cm, flow rate 1.5 mL/
min) using MeOH as the eluent, yielding 133 fractions of 15 mL
that were grouped by TLC into twelve major fractions (A–N).
Fraction B (1 g, 810–1170 mL) was partitioned between n-BuOH
and H2O, to afford a n-BuOH residue (780 mg) that was purified
by RP-HPLC with MeOH–H2O (2:3) as the eluent to give pure
phlinoside B (9 mg, tR = 13 min), compound 5, (1.1 mg, tR =
+
+
"
2α,15-dihydroxy-3β-(2′-hydroxy-2′-methyl-3′-acetoxy)-butanoyl-
oxy-6α,18-diacetoxy-4α,17-epoxy-clerodan-11,16-lactone
(3):
colorless amorphous powder; [α]2D5 − 5.7 (c 0.11, MeOH); H and
1
13
+
"
C NMR data, see l Table 1; ESI MS m/z 665 [M + Na] , 605 [M +
Na – 60]+, 486 [M + Na – 176]+; HR ESIMS m/z [M + H]+ 643.2899
(calcd. for C31H47O14 643.2966).
3β,14S,15-trihydroxy-6α,18-diacetoxy-4α,17-epoxy-clerodan-
11,16-lactone (4): colorless amorphous powder; [α]2D5 + 2.6 (c
Faiella L et al. Diterpenes and Phenylpropanoids… Planta Med 2013; 79: 1341–1347

1346 Original Papers
0.13, MeOH); 1H and 13C NMR data, see l Table 1; ESI MS m/z 507
[M + Na]+, 447 [M + Na – 60]+, 387 [M + Na – 60–60]+; HR ESIMS
m/z 485.2423 [M + H]+ (calcd. for C24H37O10 485.2387).
control samples containing equal amounts of vehicle (DMSO). To
exclude any interference of test compounds with the tetrazolium
salt-based assay, cell growth inhibition was randomly verified al-
so by cytometric count (trypan blue exclusion test). PEITC was
used as a positive control and showed an IC50 of 15 µM.
"
β-(3,4-dihydroxyphenyl)ethyl-O-β-D-xylopyranosyl-(1 → 2)-α-L-
rhamnopyranosyl-(1 → 3)-6-O‑t-caffeoyl-β-D-glucopyranoside (5):
brown amorphous powder; [α]2D5 − 10.7 (c 0.5, MeOH); UV (MeOH)
max (log ε) 220 sh (3.88), 288 sh (3.95), 330 (4.09) nm; 1H and 13
NMR data, see l Table 2; ESI MS m/z 755 [M – H] ,623 [M – H –
132]−, 593 [M – H – 162]−, 779 [M + Na]+, 647 [M + Na – 132]+,
501 [M + Na – 132–146]+; HR ESIMS m/z 757.2637 [M + H]+ (calcd.
for C34H45O19 757.2555).
λ
C
Statistical analysis
Data reported are the mean values SD of at least three experi-
ments performed in duplicate.
−
"
Supporting information
NMR spectra of compounds 1–5 are available as Supporting In-
formation. This material is available online at http://www.
thieme-connect.de/ejournals/toc/plantamedica.
Acid hydrolysis of compound 5
A solution of compound 5 (2.0 mg) in HCl 1 N (1 mL) was stirred at
80°C in a stoppered reaction vial for 4 h. After cooling, the solu-
tion was evaporated under a stream of N2. The residue was dis-
solved in 1-(trimethylsilyl)imidazole and pyridine (0.2 mL), and
the solution was stirred at 60°C for 5 min. After drying the solu-
tion, the residue was partitioned between water and CHCl3. The
CHCl3 layer was analyzed by GC using a I-Chirasil-Val column
(0.32 mm × 25 m). Temperatures of the injector and detector
were 200°C for both. A temperature gradient system was used
for the oven, starting at 100°C for 1 min and increasing up to
180°C at a rate of 5°C/min. Peaks of the hydrolysate were de-
tected by comparison with retention times of authentic samples
of L-rhamnose, D-xylose, and D-glucose (Sigma Aldrich) after
treatment with 1-(trimethylsilyl)imidazole in pyridine.
Conflict of Interest
!
The authors declare no conflict of interest.
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