Phytochemistry 154 (2018) 39–46

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Anti-inflammatory and antiproliferative diterpenoids from Plectranthus T

scutellarioides
Sylvian Crettona, Noémie Sarauxa, Aymeric Monteilliera, Davide Righia, Laurence Marcourta,
Grégory Genta-Jouveb, Jean-Luc Wolfendera, Muriel Cuendeta, Philippe Christena,∗
a
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland
b
C-TAC, UMR 8638 CNRS, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie de Paris, Avenue de l’Observatoire 4, 75006 Paris, France

A R T I C LE I N FO A B S T R A C T

Keywords: Chemical investigation of the dichloromethane extract of the aerial parts of Plectranthus scutellarioides led to the
Plectranthus scutellarioides isolation and characterization of 10 diterpenoids with an abietane skeleton and one cembrane-type diterpenoid.
Lamiaceae Among them, six have not yet been described in the literature. Their structures were established by 1D and 2D
Abietane diterpene NMR, UV and IR spectroscopy, and HRESIMS. The relative configuration was determined by Gauge-Independent
Cembrane diterpene
Atomic Orbital NMR chemical shift calculations supported by the advanced statistical method DP4 plus and
NF-κB inhibitory activity
MM-CSCs antiproliferative activity
further confirmed by electronic circular dichroism. The isolated constituents were evaluated for their in vitro NF-
κB inhibitory activity, as well as for their cytotoxic effects in human multiple myeloma cancer stem cells and
RPMI 8226 tumor cell line. Coleon O, coleon G, lanugone K and 6-acetylfredericone B showed the highest
inhibitory effect against NF-κB, displaying IC50 of 11.2, 11.0, 4.5 and 9.7 μM, respectively. Coleon O exhibited
also a significant activity towards human multiple myeloma cancer stem cells and RPMI 8226 cells with IC50 of
9.2 and 8.4 μM, respectively.

1. Introduction However, as yet, no psychoactive constituent has been found.

The genus Plectranthus L’Hér. (Lamiaceae) comprises about 300
species distributed in Tropical Africa, Asia and Australia. Some species
such as Plectranthus amboinicus, P. laxiflorus and P. barbatus are well
documented for their ethnomedicinal uses (Brito et al., 2018; Lukhoba
et al., 2006). Phytochemical studies on this genus report mainly the
presence of diterpenoids with abietane or labdane skeletons. Mono-
terpenoids, sesquiterpenoids and phenolics have also been described in
different species of Plectranthus (Lukhoba et al., 2006). However, very
few phytochemical investigations were performed on P. scutellarioides
(L.) R.Br. (syn. Coleus pumilus or Coleus blumei) (www.theplantlist.org,
2018). In spite of its traditional use for the treatment of various ail-
ments (Bourdy and Walter, 1992; Roosita et al., 2008; Waruruai et al.,
2011) only rosmarinic acid (Razzaque and Ellis, 1977), quercetin
(Moektiwardoyo et al., 2011), five abietane diterpenes derivatives co-
leon O (Devriese et al., 1988), 2,16-diacetyl derivative of
2,6,11,12,14,16,17-heptahydroxy-5,8,11,13-abietatetraen-7-one
(Ragasa et al., 2001) and spiroscutelones A-C (Ito et al., 2018) were
identified in this plant. In addition to its ethnomedicinal use as a
treatment against several inflammatory disorders, P. scutellarioides is
used as hallucinogen in the region of Oaxaca, Mexico (Schultes, 1984).
In the present study, a bioactivity-guided isolation using a NF-κB
assay was performed to support its traditional use as an anti-in-
flammatory plant and to gain knowledge on the phytochemical com-
position of this plant. Eleven terpenoids were isolated and characterized
from the aerial parts of P. scutellarioides. Among these, six compounds
(1–6) are undescribed phytochemicals (Fig. 1). The NF-κB inhibitory
activity of the isolated compounds as well as their cytotoxic effects in
human multiple myeloma cancer stem cells (MM-CSCs) and RPMI 8226
multiple myeloma plasma cells were determined.
2. Results and discussion
The crude dichloromethane extract of the aerial parts of P. scu-
tellarioides was fractionated using flash chromatography. The resulting
fractions were tested for their NF-κB inhibitory activity (Fig. S1).
Fraction F11 and those possessing higher than 40% inhibition were
purified by semi-preparative HPLC yielding six previously unreported
(1–6) and five (7–11) known compounds.
Compound 1 was obtained as a brownish oil, and its molecular
formula C20H26O7 was determined by HRESIMS from the ion m/z
379.1753 [M + H]+. The IR spectrum showed absorption bands

∗
Corresponding author.
E-mail address: philippe.christen@unige.ch (P. Christen).

https://doi.org/10.1016/j.phytochem.2018.06.012
Received 22 March 2018; Received in revised form 14 June 2018; Accepted 18 June 2018
Available online 28 June 2018
0031-9422/ © 2018 Elsevier Ltd. All rights reserved.
S. Cretton et al.
Fig. 1. Structures of the isolated compounds from Plectranthus scutellarioides.
attributable to hydroxy (3349 and 2931 cm-1) and ketone (1703 cm−1)
groups. Analysis of the 1D NMR data (Tables 1 and 2), HSQC and HMBC
spectra of 1 revealed 20 carbon signals corresponding to four methyls
C-17 to C-20 (δC 23.3, 22.9, 29.1 and 21.8, respectively), three me-
thylenes C-1, C-2 and C-15 (δC 37.1, 34.7 and 33.3, respectively), four
methines C-5 to C-7 and C-16 (δC 48.5, 69.4, 67.1 and 67.8, respec-
tively), and nine quaternary carbons, C-3, C-4 and C-8 to C-14 (δC
220.4, 48.0, 141.4, 147.4, 38.8, 184.7, 156.0, 118.5 and 188.9, re-
spectively). Considering these data and the diterpenoid fredericone B
(10) isolated from the same plant, compound 1 could be assigned as an
abietane quinone diterpenoid with the skeleton abieta-8,12-dien-
3,11,14-trione. Cross peaks between H-15/H-16 and H-16/H-17 in a
COSY experiment, and key HMBC correlations between H-15 (δH 2.55
and 2.67) and C-16, C-17, C-12, C-13 and C-14 allowed positioning a 2-
hydroxypropyl sidechain in C-13 (Fig. 2). The chemical shift of the two
methines C-6 and C-7 (δC 69.4 and 67.1, respectively) indicated the
presence of two hydroxy groups in these positions. The ROESY corre-
lations from H-5 to Me-19, H-1α, and H-2α below the plane of the
molecule and from Me-20 to H-1β and Me-18 above the plane of the
molecule allowed positioning these protons in α and β orientation, re-
spectively (Fig. 3). The ROESY correlation from H-6 to Me-18 and Me-
19 as well as the small coupling constant between H-6 and H-5 (1.5 Hz)
and H-6 and H-7 (2.8 Hz) indicated that H-6 is in a pseudo-equatorial
position and thus in an α-orientation. The lack of correlation between
H-7 and H-5 indicated that H-7 is β-oriented and thus that the hydroxy
group attached to C-7 is α-oriented (Fig. 3). Moreover, the NMR data
regarding those positions are in good agreement with the ones from
lanugone K (9) (Schmid et al., 1982). The relative configuration at C-16
was assigned by calculation of the Smith and Goodman DP4 probability
40
Phytochemistry 154 (2018) 39–46
(Smith and Goodman, 2010). Comparison of the experimental and
theoretical chemical shifts indicated a 16S* configuration with a 95.8%
probability (see supporting information). The absolute configuration of
1 was established by comparison of experimental and calculated ECD
spectra. A good agreement was observed between both experimental
and theoretical ECD spectra (Fig. 4). Compound 1 was thus identified as
the 13-isopropanol derivative of (5R,10S)-6S,7S,12-trihydroxy-8,12-
abietadien-3,11,14-trione and named scutellarioidone A.
The HRESIMS of compound 2 exhibited an ion at m/z 449.2170 [M
+ H]+ (calcd for C24H33O8, 449.2170), corresponding to the molecular
formula C24H32O8. Data provided by 1H and 13C NMR were compared
to those of lophanthoidin B (Yunlong et al., 1989) and the difference
between the two compounds was an acetoxy group in C-6 for 2 instead
of C-7 in lophanthoidin B. This was confirmed by the HMBC correlation
from H-6 (δH 5.48) and the methyl protons at δH 2.02 to the carbonyl
ester carbon at δC 171.9. The ROESY correlations from H-5 to H-1α, H-
3α and Me-19, and from H-6 to Me-19 allowed positioning all these
protons in α-orientation. The stereochemistry in C-15 was not pre-
viously determined. Therefore, a comparison of the experimental 13C
NMR chemical shifts was performed. According to the DP4 probability,
a 15S* configuration was established (85.4%). The absolute config-
uration was assigned by comparison of experimental ECD spectrum
with calculated ECD spectra (Fig. 4). Compound 2 was thus identified
as the 13-propyl acetate derivative of (5R,10S)-6S-acetoxy,7S,12-dihy-
droxy-8,12-abietadien-11,14-dione and named scutellarioidone B.
The NMR data of compound 3 were very similar to those of com-
pound 11. The molecular weight deduced from the HRESIMS ion at m/z
461.1808 [M + H]+ (calcd for C24H29O9, 461.1806) indicated a dif-
ference of 18 Da. This could be explained by an additional cyclisation in
 S. Cretton et al.

Table 1
1
H NMR data of compounds 1–6 (600 MHz, in CD3OD or *DMSO‑d6, δ in ppm, J in Hz).
Position 1 2 3 3* 4 5 6

1 β 2.66, m β 2.69, dt (13.1, 3.8) β 3.96, dd (14.1, 8.6) β 3.83, dd (13.9, 8.5) β 2.73, dd (12.3, 7.2) β 2.42, dt (12.7, 3.7)
α 1.84, ddd (11.5, 10.6, 5.4) α 1.22, m α 1.44, dd (14.1, 8.1) α 1.33, dd (13.9, 7.8) α 1.48, td (12.3, 7.1) α 1.58, td (12.7, 3.8)
2 α 2.74, ddd (15.5, 10.6, 5.2) β 1.88, qt (13.4, 3.6) 5.38, qd (8.6, 8.1, 6.7, 2.3) α 5.25, qd (8.5, 7.8, 6.4, 2.2) β 2.26, td (19.7, 12.3, 5.3) β 2.02, m 2.98, dd (16.3, 6.5)
β 2.45, ddd (15.5, 9.0, 5.4) α 1.58, dp (13.4, 3.6) α 2.06, dd (19.7, 6.7) α 1.43, d (13.9) 2.90, dd (16.3, 8.0)
3 β 1.48, dt (13.6, 3.6) β 2.40, dd (15.8, 6.7) β 2.34, dd (15.9, 6.4) β 2.60, m 5.29, t (8.0, 6.5)
α 1.30, td (13.6, 3.6) α 1.69, dd (15.8, 2.3) α 1.61, dd (15.9, 2.2)
5 α 2.33, s 1.76, s 2.50, t (1.5) α 2.36, s 2.82, dd (14.9, 5.7)
2.62, dd (14.9, 2.7)
6 α 4.10, dd (2.8, 1.3) α 5.48, dd (2.3, 1.2) 5.60, dt (5.3, 1.5) α 4.38, dt (4.2, 1.5) 5.21, dt (5.7, 2.7,1.3)
7 β 4.69, d (2.8) β 4.54, d (2.3) α 2.82, dt (21.2, 1.5) β 2.81, d (19.7) 7.30, q (1.5)
β 2.75, dd (21.2, 5.3) α 2.60, m
9 2.48, m
2.45, m
10 2.62, m
2.51, m

41
11 5.61, t (7.0)
13 5.30, t (7.2)
14 2.52, m
2.39, dd (14.2, 7.5)
15 2.67, m 3.39, q (7.7, 7.2) 3.98, tt (9.2, 7.8, 5.3, 4.1) 3.96, qd (9.2, 7.9, 5.4, 4.3) 2.47, m 2.49, td (7.0, 2.2)
2.55, dd (12.9, 6.3)
16 4.00, q (6.3) 4.31, dd (10.4, 7.2) 4.49, dd (11.0, 4.1) a 4.42, dd (10.9, 4.2) 1.78, p (6.7) 1.80, q (7.0) 1.61, s
4.24, dd (10.4, 7.7) 4.20, dd (11.0, 7.8) b 4.11, dd (10.9, 7.9)
17 1.15, d (6.3) 1.22, d (7.2) 4.76, t (9.2) a 4.77, t (9.2) 4.03, t (6.5) 4.04, t (7.0) 1.73, s
4.62, dd (9.2, 5.3) b 4.57, dd (9.2, 5.4)
18 β 1.35, s 1.06, s 1.46, s 1.39, s 1.65, s a 5.05, s 4.68, d (12.2)
b 5.01, s 4.54, d (12.2)
19 α 1.19, s 1.03, s 1.52, s 1.45, s 1.67, s α 1.10, d (7.2)
20 β 1.47, s 1.62, s 1.62, s 1.53, s 1.42, s β 1.36, s 4.82, d (12.2)
4.68, d (12.2)
Ac-2 2.00, s 1.95, s
Ac-6 2.02, s 1.97, s
Ac-13 1.99, s
Ac-16 2.02, s 1.99, s
Ac-17 1.98, s 2.01, s 2.02, s
Ac-18 2.07, s
Ac-20 2.05, s
Phytochemistry 154 (2018) 39–46
S. Cretton et al. Phytochemistry 154 (2018) 39–46

Table 2
13
C NMR data of compounds 1–6 (151 MHz, in CD3OD or *DMSO‑d6, δ in ppm).
Position 1 2 3 3* 4 5 6

1 37.1, CH2 39.5, CH2 35.1, CH2 33.5, CH2 35.5, CH2 34.2, CH2 127.1, C
2 34.7, CH2 20.0, CH2 69.7, CH 67.4, CH 31.1, CH2 29.7, CH2 32.1, CH2
3 220.4, C 43.5, CH2 43.4, CH2 41.7, CH2 128.8, C 39.5, CH 137.5, CH
4 48,0 CH 34.6, C 37.0, C 35.6, C 124.6, C 153.4, C 127.9, C
5 48.5, C 48.6, CH 143.1, C 142.9, C 48.3, CH 47.5, CH 36.8, CH2
a a
6 69.4, CH 72.0, CH 68.2, CH 65.5, CH 82.3, CH
a
7 67.1, CH 64.6, CH 183.4, C 32.1, CH2 35.8, CH2 152.1, CH
a
8 141.4, C 140.6, C 107.1, C 141.4, C 142.2, C 134.0, C
a
9 147.4, C 148.9, C 138.9, C 146.4, C 146.5, C 25.5, CH2
10 38.8, C 40.2, C 42.2, C 40.7, C 37.7, C 40.2, C 24.4, CH2
a a a
11 184.7, C 184.5, C 184.1, C 135.4, CH
a
12 156.0, C 155.0, C 158.2, C 155.3, C 155.1, C 135.4, C
13 118.5, C 121.1, C 112.1, C 111.1, C 119.7, C 119.6, C 76.2, CH
a
14 188.9, C 188.0, C 153.1, C 188.8, C 189.5, C 38.1, CH2
15 33.3, CH2 30.5, CH 41.6, CH 39.7, CH 20.1, CH2 20.2, CH2 130.9, C
16 67.8, CH 67.6, CH2 65.3, CH2 63.5, CH2 28.1, CH2 28.2, CH2 20.8, CH3
17 23.3, CH3 15.3, CH3 77.5, CH2 75.7, CH2 65.5, CH2 65.5, CH2 21.2, CH3
18 22.9, CH3 23.9, CH3 28.9, CH3 28.2, CH3 14.9, CH3 109.2, CH2 63.8, CH2
19 29.1, CH3 33.8, CH3 29.9, CH3 29.1, CH3 19.2, CH3 21.1, CH3 175.9, C
20 21.8, CH3 22.2, CH3 24.9, CH3 24.2, CH3 19.9, CH3 20.5, CH3 60.2, CH2
Ac-2 21.3, CH3 21.2, CH3
172.5, C 170.1, C
Ac-6 21.3, CH3 21.2, CH3
171.9, C 172.3, C
Ac-13 172.1, C
21.1, CH3
Ac-16 20.7, CH3 20.8, CH3
172.9, C 170.7, C
Ac-17 20.8, CH3 20.8, CH3 20.9, CH3
173.0, C 173.0, C 173.1, C
Ac-18 172.8, C 20.8, CH3
AC-20 172.5, C
20.8, CH3

a
Signal too weak to be measured.

Fig. 2. Key COSY (red bold line) and HMBC (blue arrows) correlations of compounds 1, 3 and 6. (For interpretation of the references to colour in this figure legend,
the reader is referred to the Web version of this article.)

3 to form a fourth cycle linked to the abietane skeleton in C-12 and C-
13. Indeed, the HMBC correlations from H-17 (δH 4.76 and 4.62) to C-
12 (δC 158.2) and C-13 (δC 112.1), and a COSY correlation between H-
15/H-17 suggested a cyclopentane ring (Fig. 2). Moreover, the chemical
shifts of C-17 (δC 77.5) and C-12 indicated an ether bridge between
these two carbons. In the HMBC spectrum, correlations between H-2
(δH 5.37) and carbonyl group of Ac-2 (δC 172.5), and between H-16 (δH
4.20) and Ac-16 (δC 172.9) allowed to localize unequivocally these two
acetoxy groups on C-2 and C-16. The ROESY correlations observed from
H-2 to H-1β, H-3β and Me-20 and from H-3β to Me-20 and Me-18 al-
lowed positioning the acetate in C-2 in α-orientation. Due to the ab-
sence of ROESY correlation between the chiral center at C-15 and the
other part of the molecule, the relative configuration was determined
by comparison between the theoretical and experimental chemical
shifts. A 15S* configuration was obtained with 71% probability. The
absolute configuration was elucidated by ECD. The good agreement
between the experimental and calculated spectra (Fig. 4) led to the
conclusion that 3 is ((2S,8S,11bR)-2-acetoxy-5,7,11-trihydroxy-
4,4,11b-trimethyl-6-oxo-1,2,3,4,6,8,9,11b-octahydrophenanthro[3,2-
b]furan-8-yl)methyl acetate and named scutellarioidone C.
The NMR spectra of compound 4 exhibited very strong similarities
to those of fredericone B (10). Both compounds shared the same 3,4-
dimethyl,12-hydroxy-3,8,12-abietatrien-11,14-dione skeleton with a
13-propyl acetate moiety. The only difference lies on the acetylation of
the hydroxy group in C-6 for compound 4 showed in the HMBC spec-
trum displaying a correlation between H-6 at δH 5.60 and the carbonyl
carbon at δC 172.3 (Ac-6). The HRESIMS of 4 showed a sodium adduct
at m/z 453.1885 [M + Na]+ (calcd for C24H30O7Na, 453.1889), in-
dicating a molecular formula as C24H30O7, which is in agreement with
the 6-acetyl derivative of fredericone B elucidated by NMR spectro-
scopy.
Compound 5 was isolated as a brownish oil, and its molecular for-
mula was established as C22H28O6 based on the HRESIMS ion at m/z
389.1954 [M + H]+. The 1H and 13C NMR spectroscopic data of 5

42
S. Cretton et al. Phytochemistry 154 (2018) 39–46

Fig. 3. 3D structures of compounds 1, 2 and 5 and key ROESY (black arrow) correlations.

Fig. 4. Experimental and TDDFT predicted ECD spectra of 1, 2 and 3.

(Tables 1 and 2) are similar to those of the isomer fredericone B (10). these protons are in β-orientation. As for 1 and for the same reasons, H-
The two differences lie in the presence of an exomethylene group (δH 6 was positioned in an equatorial position and in α-orientation (Fig. 3).
5.01 and 5.05, δC 109.2) linked to C-4 in 5, instead of a methyl group in Therefore, compound 5 was identified as the 13-propyl acetate deri-
10, and the absence of a double bond between C-3 and C-4 in 5. The vative of 3α-methyl,4-exomethylene,6β,12-dihydroxy-8,12-abietadien-
ROESY correlations from H-5 to H-1α, Me-19 and H-7α suggested that 11,14-dione and named scutellarioidone D.
all these protons are α-oriented whereas the dipolar correlations from Compound 6 was isolated as an optically active [a]22 D - 42 (c 0.1,
Me-20 to H-2β and H-18a, and from H-18b to H-3β indicated that all MeOH) pink oil. The molecular formula C26H34O8 was established by
HRESIMS from the sodium adduct at m/z 497.2147. In addition, in the
mass spectrum of 6, three fragment ions at m/z 415.2120 (C24H31O6),
Table 3 m/z 355.1906 (C22H27O4) and m/z 295.1695 (C20H23O2) suggested the
NF-κB inhibitory activity and antiproliferative activity on MM-CSCs and RPMI presence of three acetoxy groups into the molecule. IR spectroscopy
8226 cells of the compounds isolated from Plectranthus scutellarioides. indicated the presence of carbonyl groups (1732, 1649 cm−1) in the
Compound NF-κB MM-CSCs RPMI 8226 molecule. Analysis of the 1H, 13C and HSQC NMR data revealed signals
for five methyls (δH 1.61, 1.73, 1.99, 2.05 and 2.07), seven methylenes
IC50 [μM]
(δC 24.4, 25.5, 32.1, 36.8, 38.1, 60.2 and 63.8), two sp3 carbons
1 > 100 > 100 > 100 bearing an oxygen atom (δC 76.2 and 82.3), three olefinic protons (δH
2 42.0 ± 4.8 > 100 > 100 5.29, 5.61 and 7.30), five sp2 quaternary carbons (δC 127.1, 127.9,
3 > 100 > 100 > 100 130.9, 134.0 and 135.4) and four carbonyls (δC 172.1, 172.5, 172.8 and
4 9.7 ± 1.0 17.6 ± 0.7 21.6 ± 0.4
175.9). 2D experiments (COSY and HMBC) allowed to interconnect the
5 39.0 ± 5.0 > 100 > 100
6 50.3 ± 1.7 > 100 > 100
different elements and identify a cembranoid diterpene possessing an
7 11.2 ± 3.6 9.2 ± 0.4 8.4 ± 0.3 α,β-unsaturated butenolactone moiety, an isobutylene unit attached in
8 11.0 ± 2.4 37.4 ± 1.1 38.4 ± 2.1 C-1, and three acetoxy groups linked to C-13, C-18 and C-20 (Fig. 2).
9 4.5 ± 1.7 > 100 > 100 The ROESY correlations between H-3 and H-5 from one side, and be-
10 21.5 ± 2.2 > 100 > 100
tween H-2 and H-18 from the other side imply a configuration Z for the
11 > 100 > 100 > 100
Parthenolide 0.55 ± 0.01 double bond between C-3 and C-4. In contrast, the NOE observed be-
Bortezomib 0.008 0.003 tween H-11 and H-13, and between H-20 and H-10 indicate a

43
S. Cretton et al.
configuration E for the double bond between C-11 and C-12. To de-
termine the relative stereochemistry, density functional theory (DFT)
calculation were undertaken to take advantage of the DP4 approach.
According to the calculation, a 99.9% probability was obtained in favor
of a (3Z*,6S*,7Z*,11E*,13R*) structure. Therefore, compound 6 was
fully elucidated as (3Z*,6S*,7Z*,11E*,13R*)-1-(2-methylprop-1-ene)-
13,18,20-triacetoxycembra-1(15),3,7,11-tetraene-6,19-olide and
named scutellarioidolide A.
The known compounds were identified based on their spectroscopic
data and by comparison with the literature. Coleon O (7) (Arihara et al.,
1975) and coleon G (8) (Moir et al., 1973) were previously isolated
from Coleus somaliensis. Lanugone K (9) was formerly identified in P.
lanuginosus (Schmid et al., 1982), fredericone B (10) in Coleus fredericii
(Zhu et al., 1988), and the 2,16-diacetyl derivative of
2,6,11,12,14,16,17-heptahydroxy-5,8,11,13-abietatetraen-7-one (11)
in Coleus blumei (Ragasa et al., 2001).
The 11 isolated compounds were tested for their inhibitory prop-
erties against TNFα-induced NF-κB activation and for their anti-
proliferative activity in MM-CSCs and RPMI 8226 cells as abietane di-
terpenes have previously shown antiproliferative activity in human
cancer cells (Gonzalez, 2015; Johnson, 2011). The data obtained are
summarized in Table 3. Coleons O and G (7 and 8, respectively), la-
nugone K (9) and 6-acetylfredericone B (4) showed the highest in-
hibitory activity against NF-κB with IC50 ranging from 4.5 to 11.2 μM. It
is noteworthy that compounds 7, 8, and 9 share the same 7α-acetoxy-
6β,12α-dihydroxy-8-abietaen-11,14-dione skeleton with a methylated
spiro-cyclopropane structure in C-13. As far as we know, this scaffold
has never been reported for its NF-κB inhibition and can be of interest
for the development of new inhibitors. Compound 7 also exerted the
strongest activity on the growth of MM-CSCs and RPMI 8226 cells (IC50
9.2 and 8.4 μM, respectively), whereas the activity of 8 and 4 were
slightly lower (IC50 17–40 μM). Given the strong dependence of MM
tumors on NF-κB pathway activation (Demchenko et al., 2014;
Demchenko and Kuehl, 2010), inhibition of NF-κB pathways by the
abietane diterpenes 7, 8, and 4 may explain their antiproliferative ac-
tivity in MM cancer cells. Moreover, the activity towards CSCs, which
are normally more resistant than plasma cells, was similar to the one
against RPMI 8226 cells.
3. Concluding remarks
The phytochemical study of the aerial parts of P. scutellarioides re-
sulted in the isolation of one new cembranoid and 10 abietane di-
terpenes. Among the latter, compounds 7, 8, 9 and 4 exhibited sig-
nificant NF-κB inhibitory activity, and to a lesser extend a cytotoxic
effect against MM-CSCs and RPMI 8226 cells. These secondary meta-
bolites may explain the traditional use of P. scutellarioides preparations
to treat diverse inflammatory disorders.
4. Experimental
4.1. General experimental procedures
Optical rotations were measured on a JASCO P-1030 (Easton, MD,
USA) polarimeter. The ECD spectra were recorded on a JASCO J-815
CD spectrometer. UV spectra were recorded on a Perkin-Elmer Lambda-
25 UV–vis spectrophotometer (Wellesley, MA, USA). IR spectra were
measured on a Perkin-Elmer Spectrum 100 spectrometer. NMR spectra
were recorded on a Bruker Avance III HD 600 MHz NMR spectrometer
equipped with a QCI 5 mm Cryoprobe and a SampleJet automated
sample changer (Bruker BioSpin, Rheinstetten, Germany). Chemical
shifts are reported in parts per million (δ) using the residual CD3OD
signals (δH 3.31; δC 49.0) or DMSO‑d6 signal (δH 2.50; δC 39.5) as in-
ternal standards for 1H and 13C NMR, respectively. HRMS spectra were
obtained on a Q Exactive Plus Hybrid quadrupole-orbitrap mass spec-
trometer (Thermo Scientific, Waltham, MA, USA) using electrospray in
44
Phytochemistry 154 (2018) 39–46
the positive mode. The spray voltage was set to 3.5 kV; the sheath gas
flow rate (N2) to 50 units; the capillary temperature to 320 °C; the S lens
RF level to 50; and the probe heater temperature to 425 °C. UHPLC was
performed on an Ultimate 3000 UHPLC System (Thermo Scientific).
The separation was performed on an Acquity BEH C18 UPLC column
(100 × 2.1 mm i.d.; 1.7 μm, Waters), using a gradient (MeCN and H2O
both containing 0.1% formic acid) of 5–98% MeCN in 6 min, followed
by a washing step with 98% MeCN for 2 min. After the washing step,
the column was equilibrated with 5% MeCN for 3 min before the next
injection. The flow rate was set to 0.4 mL/min, the temperature to
40 °C, and the injection volume was 1 μL. Fractionation was performed
on a Puriflash 4100 preparative chromatographic system (Interchim,
Montluçon, France) equipped with a quaternary pump, a PDA detector
and a fraction collector. Semi-preparative chromatography was per-
formed on an Armen Spot System (Saint-Avé, France). The control of
fractions was carried out by an Acquity UPLC System (Waters) equipped
with an Acquity PDA detector and connected to a Quattro Micro triple
quadrupole mass spectrometer (Waters) with an ESI source operating in
positive mode.
4.2. ECD computational details
All calculations were performed using the Gaussian 16 program
(Frisch et al., 2016). After a conformational analysis using the GMMX
package with MMFF92 force field (ΔE < 1 kcal/mol), geometry op-
timization was achieved on the most stable conformer using density
function theory (DFT) with the B3LYP functional and the 6-31G(d) basis
set in the gas-phase. Vibrational analysis was completed at the same
level to confirm a minimum. NMR prediction was performed using the
mPW1PW91/6-311 + g(2d,p) level, while rotational strengths were
evaluated using the B3LYP/6-31g(d) method for 30 excited states. ECD
curves were constructed on the basis of rotatory strength dipole velocity
(Rvel) using SpecDis v1.61 (Bruhn et al., 2013).
4.3. Plant material
The aerial parts of Plectranthus scutellarioides (L.) R.Br. (Lamiaceae)
were collected in July 2015 in the Botanical Garden of Geneva and
identified by Mr Didier Roguet. A voucher specimen (No. 37001) was
deposited in the Herbarium of the Botanic Garden of Geneva.
4.4. Extraction and isolation
Fresh aerial parts (0.8 kg) were soaked with 4 L of CH2Cl2 at room
temperature for five minutes to obtain a terpenoid-enriched extract
devoid of other lipophilic compounds such as waxes and chlorophylls
(Siebert, 2004). After filtration, the extract was evaporated to dryness.
The residue (0.93 g) was mixed with 4 g of Celite 577 (Fluka, AG,
Switzerland) and introduced into a cartridge for a dry load injection.
Fractionation of the extract was performed using two flash chromato-
graphy columns connected in series (PF-C18HQ/120 g, 15 μm C18, In-
terchim). Fractionation was carried out with the mobile phase H2O and
MeCN both containing 0.1% formic acid in an optimized gradient
mode: 35–70% MeCN in 50 min, and 70–100% in 10 min. The flow rate
was set to 16 mL/min and the UV detection was simultaneously per-
formed at 220, 254, and 360 nm. The separation yielded 120 fractions,
which were analyzed individually by UHPLC-PDA-MS, and gathered in
24 fractions according to their chromatographic profiles. Fractions 11,
14–16 and 20–24 were selected for further purification. The final
fractionation steps were performed by semi-preparative HPLC using a
Kinetex Axia Core-Shell C18 column (5 μm, 250 × 21.2 mm; Phenom-
enex Torrance, CA, USA) using H2O/MeCN/0.1% formic acid as sol-
vents for an isocratic elution. The flow rate was set to 20 mL/min and
UV absorbance was at 280 nm. Fraction 11 (15.2 mg) yielded 1 (1.4 mg)
(25% MeCN); fraction 14 (12.0 mg) yielded 7 (2.8 mg) (35% MeCN);
fraction 15 (7.1 mg) yielded 8 (0.5 mg) (35% MeCN); fraction 16
S. Cretton et al.
(6.2 mg) yielded 9 (0.6 mg) (35% MeCN), fractions 20 (16.0 mg)
yielded 6 (3.1 mg) (45% MeCN), fraction 21 (13.0 mg) yielded 10
(0.8 mg) and 5 (1.2 mg) (45% MeCN), fraction 22 (13.0 mg) yielded 2
(1.0 mg) (45% MeCN), fraction 23 (12.0 mg) yielded 3 (0.7 mg), frac-
tion 24 (20.0 mg) yielded 11 (1.2 mg) and 4 (1.5 mg) (48% MeCN).
4.4.1. Scutellarioidone A (1)
D + 48 (c 0.1, MeOH); ECD (c 0.05, MeCN) λmax
Brownish oil; [a]22
nm (Δε) 210 (1.4), 224 (−1.2), 260 (5.9), 344 (−1.3); UV (MeCN) λmax
219, 270 nm; IR (CHCl3) νmax 3349, 2931, 1703, 1263, 1021 cm−1; 1H
and 13C NMR, see Tables 1 and 2; HRESIMS m/z 379.1753 [M + H]+
(calcd for C20H27O7, 379.1752).
4.4.2. Scutellarioidone B (2)
D - 17 (c 0.1, MeOH); ECD (c 0.05, MeCN) λmax (Δε)
Brownish oil; [a]22
224 (−1.8), 295 (4.26), 403 (−2.01); UV (MeCN) λmax 225 nm; IR
(CHCl3) νmax 3362, 2930, 1731, 1376, 1243 cm−1; 1H and 13C NMR, see
Tables 1 and 2; HRESIMS m/z 449.2170 [M + H]+ (calcd for C24H33O8,
449.2170).
4.4.3. Scutellarioidone C (3)
Violet oil; ECD (c 0.05, MeCN) λmax (Δε) 201 (8.74), 273 (5.07), 298
(−2.67); UV (MeCN) λmax 217, 265, 328, 388 nm; IR (CHCl3) νmax
2954, 1739, 1369, 1223 cm−1; 1H and 13C NMR, see Tables 1 and 2;
HRESIMS m/z 461.1808 [M + H]+ (calcd for C24H29O9, 461.1806).
4.4.4. 6-Acetylfredericone B (4)
D + 83 (c 0.1, MeOH); UV (MeCN) λmax 224,
Brownish oil; [a]22
276 nm; IR (CHCl3) νmax 2946, 1737, 1369, 1231, 1036 cm−1; 1H and
13
C NMR, see Tables 1 and 2; HRESIMS m/z 453.1885 [M + Na]+
(calcd for C24H30O7Na, 453.1889).
4.4.5. Scutellarioidone D (5)
D + 65 (c 0.1, MeOH); UV (MeCN) λmax 223,
Brownish oil; [a]22
277 nm; IR (CHCl3) νmax 3378, 2930, 1726, 1493, 1254 cm−1; 1H and
13
C NMR, see Tables 1 and 2; HRESIMS m/z 389.1954 [M + H]+ (calcd
for C22H29O6, 389.1959).
4.4.6. Scutellarioidolide A (6)
D - 42 (c 0.1, MeOH); UV (MeCN) λmax 217, 270 nm; IR
Pink oil; [a]22
(CHCl3) νmax 2927, 1732, 1649, 1436, 1371, 1231 cm−1; 1H and 13C
NMR, see Tables 1 and 2; HRESIMS m/z 497.2147 [M + Na]+ (calcd
for C26H34O8Na, 497.2151).
4.5. NF-κB inhibitory activity
NF-κB inhibitory activity was assessed as previously described (Tran
et al., 2015). Briefly, HEK293/NF-κB-luc cells (Panomics, Fremont, CA,
USA) were cultured at 37 °C and 5% CO2 atmosphere in high glucose
Dulbecco's modified Eagle's medium (Thermo Fisher scientific, Wal-
tham, MA, USA) with 10% fetal bovine serum (Biowest, Nuaillé,
France), 100 U/mL penicillin, 100 μg/mL streptomycin (Thermo Fisher
scientific) and 100 μg/mL hygromycin B (Thermo Fisher scientific). The
cells were incubated for 1 h in FBS-free medium supplemented with
2.5 μM of Cell Tracker Green CMFDA (Thermo Fisher scientific), a
fluorescent probe used to measure cell viability, and reseeded in 96-
well plates (1 × 104 cells/well) overnight. Cells were then treated with
the sample or vehicle only (0.5% DMSO in culture medium) and sti-
mulated with TNFα (20 ng/mL) for 5 h. Reporter lysis buffer (Promega,
Madison, WI, USA) was added to lyse the cells and both fluorescence of
the Cell Tracker Green CMFDA and luminescence of the firefly luci-
ferase were read on a Cytation 3 plate reader (Biotek, Winooski, VT,
USA). The luminescence signal was normalized by the fluorescence
signal for each well, and quantification of the relative NF-κB activity
was performed by comparing the normalized luminescence signal of
sample-treated cells with the vehicle-treated cells. IC50 values were
45
Phytochemistry 154 (2018) 39–46
calculated using nonlinear regression (with sigmoidal dose response) in
GraphPad Prism 6.05. Each compound was tested in duplicate and three
independent experiments were performed. Parthenolide was used as
positive control.
4.6. Antiproliferative activity
Human MM-CSCs derived from the bone marrow of a multiple
myeloma patient were obtained from Celprogen (Torrance, CA, USA).
MM-CSCs were used between passage 4 and 12 for experiments. The
tumoral plasma cells RPMI 8226 were obtained from LGC standards
(Middlesex, United Kingdom). RPMI 8226 cells were used between
passage 10 and 25 for experiments. The two cell lines were cultured at
37 °C and 5% CO2 atmosphere in RPMI 1640 culture medium supple-
mented with 10% fetal bovine serum), 100 μg mL−1 penicillin and
250 μg mL−1 streptomycin. MTT and XTT assays were used to evaluate
the antiproliferative activity of the 11 compounds in MM-CSCs and
RPMI 8226 cells, respectively (Issa and Cuendet, 2017). MM-CSCs were
seeded in 96 well plates at a density of 5000 cells per well and allowed
to adhere for 24 h, whereas RPMI 8226 cells were plated at a density of
15 000 cells per well and treated immediately. MM-CSCs and RPMI
8226 were treated with increasing concentrations (0–100 μM) of com-
pounds for 72 h. 20 μL of MTT solution (5 mg/mL) or 50 μL XTT solution
(1 mg/mL) were added in each well and incubated for 2 h (MTT assay)
or 4 h (XTT assay). The media and MTT solution were aspirated and the
cells containing formazan were solubilized in 100 μL DMSO. Absor-
bance was measured at 590 nm (MTT assay) or 450 nm (XTT assay).
The percentage of cell viability was calculated as the absorbance of
each well was divided by that of the vehicle control wells (0.5% DMSO
in culture medium) and multiplied by 100. IC50 values were calculated
using GraphPad Prism 6.05. Bortezomib was used as positive control.
Notes
The authors declare no competing financial interest.
Acknowledgments
JLW is thankful to SNF for the funding that permitted the acquisi-
tion of the 600 MHz NMR instrument (SNF R'Equip grant no
316030_164095).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.phytochem.2018.06.012.
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