C28Terpenoids from Lamiaceous Plant Perovskia scrophulariifolia: Their Structures and Anti-neuroinflammatory Activity

Article abstract of DOI:10.1021/acs.orglett.0c02855

Structurally unique C28 terpenoids, perovsfolins A (1) and B (2), were isolated from the aerial parts of an Uzbek medicinal plant, Perovskia scrophulariifolia (Lamiaceae). Their chemical structures including an unprecedented 6/8/6/6/6 pentacyclic carbon skeleton with a C6-C3 ester moiety were elucidated on the basis of spectroscopic analyses aided by density functional theory calculations as well as chemical evidence. Perovsfolin B (2) exhibited an anti-neuroinflammatory activity.

Full text of DOI:10.1021/acs.orglett.0c02855

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C28 Terpenoids from Lamiaceous Plant Perovskia scrophulariifolia:
Their Structures and Anti-neuroinflammatory Activity
Naonobu Tanaka, Kanji Niwa, Seita Kajihara, Daisuke Tsuji, Kohji Itoh, Nilufar Z. Mamadalieva,
and Yoshiki Kashiwada*
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ABSTRACT: Structurally unique C28 terpenoids, perovsfolins A (1) and B (2), were isolated from the aerial parts of an Uzbek
medicinal plant, Perovskia scrophulariifolia (Lamiaceae). Their chemical structures including an unprecedented 6/8/6/6/6
pentacyclic carbon skeleton with a C6−C3 ester moiety were elucidated on the basis of spectroscopic analyses aided by density
functional theory calculations as well as chemical evidence. Perovsfolin B (2) exhibited an anti-neuroinflammatory activity.
he Perovskia plants, a small group of the Lamiaceae
Tfamily, are aromatic shrubs growing in the arid region of
Central Asia.1 Various diterpenes and related terpenoids such
as abietane diterpene glycosides,2 20-norabietane diterpenes,3
diterpene dimers,4 and a C23 terpene5 have been isolated from
P. atriplicifolia, whereas abietane and 20-norabietane diter-
penes6 and conjugates of icetexane diterpene and mono-
terpene7 have also been isolated from another species, P.
abrotanoides. The aerial parts of P. scrophulariifolia have been
used as a traditional herbal medicine to treat dermatitis and
human intestinal parasites in Uzbekistan, from which abietane
diterpenes have been reported to date.8 As part of our research
for specialized metabolites possessing unique chemical
structures from traditional herbal medicines collected around
the world including Japan,9 China,10 Mongolia,11 and
Uzbekistan,12 the aerial parts of P. scrophulariifolia have been
investigated to furnish two novel C28 terpenoids, perovsfolins
A (1) and B (2). We describe herein the isolation, structure
elucidation, and evaluation of biological activity of 1 and 2.
The MeOH extract from the aerial parts of P. scrophular-
iifolia was partitioned with EtOAc and water. The EtOAc-
soluble materials were dissolved in 90% MeOH aq. and then
partitioned with n-hexane. The defatted materials were
repeatedly separated by chromatographies to give perovsfolins
A (1, 3.7 mg) and B (2, 2.7 mg) together with a known
compound (3), which was identified as methyl rosmarinate.13
Perovsfolin A (1), a colorless amorphous solid, was optically
active {[α]D +251.1 (c 0.1, MeOH)}. UV absorptions at 249,
288, and 335 nm revealed that 1 had a conjugated system. The
1H NMR spectrum displayed the resonances due to eight
aromatic or olefinic protons, five sp3 methines, of which two
were oxygenated, three sp3 methylenes, and three tertiary
methyls, including one methoxy methyl, and two secondary
methyls (Table S1). The 13C NMR spectrum showed 38
signals including one sp3 quaternary, one oxygenated tertiary,
20 aromatic or olefinic, and three carbonyl carbon signals.
These NMR data and an ion peak at m/z 677.2360 ([M +
Na]+, calcd for C38H38O10Na+, 677.2357) obtained by the
high-resolution electrospray ionization mass spectrometry
(HRESIMS) suggested the molecular formula of 1 to be
C38H38O10.
2D NMR analysis implied that 1 consisted of two partial
structures (units A and B). The presence of one 1,2,4,5-
tetrasubstituted benzene (C-1′−C-6′) and one 1,2,4-trisub-
stituted benzene (C-10′−C-15′) was revealed by analysis of
the 1D NMR and heteronuclear multiple bond correlation
(HMBC) spectra (Figure 1), whereas the 13C resonances of C-
3′ (δC 148.5), C-4′ (δC 147.1), C-12′ (δC 147.1), and C-13′
(δC 146.3) implied the existence of hydroxy groups at these
aromatic carbons. The spin systems of H-7′/H-8′ and H2-16′/
Received: August 25, 2020
https://dx.doi.org/10.1021/acs.orglett.0c02855
© XXXX American Chemical Society
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was further supported by a rotating frame Overhauser effect
spectroscopy (ROESY) correlation for H3-16/H-5′. Thus the
planar structure of perovsfolin A (1) was assigned, as shown in
Figure 2.
The H-1/H-8′-anti and H-7′/H-8′-syn relationships of 1
were implied by the analysis of the coupling constants
3
(3JH‑1/H‑8′ = 11.2 Hz and JH‑7′/H‑8′ = 3.4 Hz). The relative
configurations were underpinned by ROESY correlations
among protons on the β-side of the molecule (H-1/H-3β
and H-1/H-7′) as well as those on the α-side (H-2α/H3-18
and H-2α/H-8′) (Figure 3). In addition, the C-11R*
configuration of 1 was assigned by a steric restriction of the
dihydrofuran ring (C-1′, C-6′, C-7′, and C-11′).
Figure 1. Selected 2D NMR correlations and partial structures (units
A and B) of perovsfolin A (1).
H-17′ were found by 1H−1H COSY analysis, and the
connectivities of C-7′ to C-1′ and of C-16′ to C-10′ were
assigned by HMBC analysis. In addition, HMBC cross-peaks
of the methoxy signal and H2-16′ to one ester carbonyl carbon
(C-18′, δC 170.4) and of H-7′ and H-17′ to the other carbonyl
carbon (C-9′, δC 174.4) suggested the connectivity of a
methoxy carbonyl group to C-17′ and the presence of an ester
linkage between C-9′ and C-17′. Therefore, the partial
structure corresponding methyl rosmarinate (C-1′−C-18′)
was assigned as unit B. Meanwhile, the interpretation of the
1H−1H COSY and HMBC spectra lead to the structures of an
α-isopropyl-α,β-unsaturated ketone moiety (C-12−C-17) and
a tetrahydronaphthalene moiety (C-1−C-10) with a geminal
dimethyl group at C-4. HMBC correlations for H-14 with C-7,
C-8, and C-9 suggested the connectivity between C-8 and C-
14. Thus the gross structure of unit A was assigned, as shown
in Figure 1.
Figure 3. Selected ROESY correlations and the relative configuration
of perovsfolin A (1). (C-9′−C-18′ and protons of methyl groups are
omitted.)
Perovsfolin B (2) was obtained as an optically active
amorphous solid {[α]D −190.7 (c 0.1, MeOH)}, and its
molecular formula was determined to be the same as 1 by
HRESIMS analysis. The 1D NMR spectra of 2 were closely
correlated with those of 1 (Table S1), suggesting that 2 was a
stereoisomer of 1. The ROESY spectrum of 2 showed
correlations similar to those of 1, implying that the relative
configurations of C-1, C-11, C-7′, and C-8′ for 2 and 1 were
the same.
The linkage between C-1 in unit A and C-8′ in unit B was
1
evident from a H−1H COSY cross-peak of H-1/H-8′ and an
HMBC correlation for H-8′/C-10 (Figure 2). Furthermore,
the connectivities among C-9, C-12, and C-6′ via an
oxygenated tertiary carbon (C-11, δC 96.7) and of C-7′ and
C-11 via an ether linkage were elucidated by HMBC cross-
peaks of H-5′ and H-7′ to C-11, taking the molecular formula
of 1 into consideration, to appear as a structure corresponding
to a 20-norabietane diterpene (C-1−C-19). This assignment
The absolute configurations of C-17′ in perovsfolins A (1)
and B (2) were confirmed as follows (Scheme 1). Methylation
of 1 and 2 with CH3I afforded permethyl derivatives (1a and
2a). Treatment of 1a and 2a with NaOH in MeOH/acetone
gave terpenoid (1b and 2b, respectively) and C6−C3 (1c and
2c, respectively) moieties in each case. The latter products,
both of which were shown to have a carboxylic acid group,
were further treated with TMS−CHN2 to furnish trimethyltan-
shinols (1d and 2d). In contrast, the methylation of methyl
rosmarinate (3) gave a rosmarinic acid permethylate (3a),
which was subsequently treated with K2CO3/MeOH to give
3b. The R configuration of 3b was confirmed by the
comparison of its specific rotation value {[α]D +11.7 (c 0.3,
CH2Cl2)} with the literature value {[α]D +10.6 (c 0.67,
CH2Cl2)}.14 The racemate of 3b was obtained according to the
procedure described in the literature.15 Thus Dess−Martin
oxidation of (+)-3b followed by reduction with NaBH4 gave
( )-3b. The separation of the enantiomers {( )-3b} on chiral
high-performance liquid chromatography (HPLC) showed the
separated peaks [tR 12.1 min for (−)-3b; 14.7 min for (+)-3b],
whereas analyses of 1d and 2d under the same conditions gave
a single peak at 14.7 min in each case. These findings clearly
indicated the absolute configuration of C-17′ in both 1 and 2
to be R.
Figure 2. Selected 2D NMR correlations and the planar structure of
perovsfolin A (1).
B
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Scheme 1. Chemical Conversions of Perovsfolins A (1) and B (2) and Methyl Rosmarinate (3)
The absolute configurations of the terpenoid moieties (1b
and 2b) were assigned by electron circular dichroism (ECD)
analysis. The ECD spectra of 1b and 2b indicated that they
were enantiomers, and the spectrum of 1b was well correlated
with the time-dependent density functional theory (TDDFT)
calculated spectrum of a possible enantiomer with the 1R, 11R,
7′R, and 8′R configurations (Figure 4). Accordingly, the 1R,
presumed to be generated by the condensation of a 20-
norabietane diterpene, dehydromiltirone,16 and methyl ros-
marinate (3) (Scheme S1), where spontaneous (nonenzy-
matic) reactions may be involved. 20-Norabietane diterpenes
are recognized as constituents of Lamiaceous plants belonging
to the genera Salvia17 and Perovskia.3,6 In contrast, rosmarinic
acid is a caffeic acid ester of tanshinol widely distributed in the
plant kingdom and shows various biological activities such as
anti-inflammatory, antioxidant, and neuroprotective activ-
ities.18 Methyl rosmarinate (3) has also been reported to be
a plant metabolite possessing an anti-inflammatory activity.19
In our search for natural products to be a potential lead for a
therapeutic agent for neuroinflammation-related diseases,11
perovsfolins A (1) and B (2) were evaluated for their anti-
inflammatory activity on microglial cells. Thus perovsfolin B
(2) demonstrated an inhibitory effect on IL-1β production
from lipopolysaccharide (LPS)-stimulated microglial cells
(EC50 38.4 μM) without cytotoxicity against microglial cells
(IC50 > 50 μM). Their antiproliferative activity against human
cancer cell lines (A549, HeLa, and MCF-7) was also evaluated,
and 2 showed cytotoxicity with IC50 values of 32.1, 26.5, and
29.6 μM, respectively. In contrast, neither anti-inflammatory
nor antiproliferative activities were found in 1.
ASSOCIATED CONTENT
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sı
* Supporting Information
Figure 4. Experimental and TDDFT calculated ECD spectra of the
terpenoid moieties (1b and 2b) of perovsfolins A (1) and B (2).
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c02855.
Experimental section and 1D and 2D NMR spectra of
perovsfolins A and B (PDF)
11R, 7′R, 8′R, and 17′R configurations of perovsfolin A (1)
and the 1S, 11S, 7′S, 8′S, and 17′R configurations of
perovsfolin B (2) were elucidated. It is noteworthy that 1
and 2 showed Cotton effects similar to those of 1b and 2b,
respectively (Figure S1).
The investigation of the aerial parts of an Uzbek medicinal
plant Perovskia scrophulariifolia resulted in the isolation of two
terpenoids, perovsfolins A (1) and B (2), possessing a C28
terpenoid moiety with an unprecedented 6/8/6/6/6 pentacy-
clic carbon skeleton. Perovsfolins A (1) and B (2) were
AUTHOR INFORMATION
■
Corresponding Author
Yoshiki Kashiwada − Graduate School of Pharmaceutical
Sciences, Tokushima University, Tokushima 770-8505, Japan;
orcid.org/0000-0002-5429-3211; Email: kasiwada@
tokushima-u.ac.jp
C
https://dx.doi.org/10.1021/acs.orglett.0c02855
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Authors
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(12) Tanaka, N.; Takaishi, Y.; Shikishima, Y.; Nakanishi, Y.; Bastow,
K.; Lee, K.-H.; Honda, G.; Ito, M.; Takeda, Y.; Kodzhimatov, O. K.;
Ashurmetov, O. J. Nat. Prod. 2004, 67, 1870−1875.
(13) Woo, E.-R.; Piao, M. S. Arch. Pharmacal Res. 2004, 27, 173−
176.
Naonobu Tanaka − Graduate School of Pharmaceutical
Sciences, Tokushima University, Tokushima 770-8505, Japan;
orcid.org/0000-0002-9281-7098
Kanji Niwa − Graduate School of Pharmaceutical Sciences,
Tokushima University, Tokushima 770-8505, Japan
Seita Kajihara − Graduate School of Pharmaceutical Sciences,
Tokushima University, Tokushima 770-8505, Japan
Daisuke Tsuji − Graduate School of Pharmaceutical Sciences,
Tokushima University, Tokushima 770-8505, Japan
Kohji Itoh − Graduate School of Pharmaceutical Sciences,
Tokushima University, Tokushima 770-8505, Japan
Nilufar Z. Mamadalieva − Institute of the Chemistry of Plant
Substances, Academy Sciences of of the Republic of Uzbekistan,
Tashkent 100170, Uzbekistan
(14) O’Malley, S. J.; Tan, K. L.; Watzke, A.; Bergman, R. G.; Ellman,
J. A. J. Am. Chem. Soc. 2005, 127, 13496−13497.
(15) Zhang, L.; Zhu, L.; Yang, J.; Luo, J.; Hong, R. J. Org. Chem.
2016, 81, 3890−3900.
(16) Lin, L. Z.; Wang, X. M.; Huang, X. L.; Huang, Y.; Yang, B. J.
Acta Pharm. Sinica 1988, 23, 273−275.
(17) Wu, Y.-B.; Ni, Z.-Y.; Shi, Q.-W.; Dong, M.; Kiyota, H.; Gu, Y.-
C.; Cong, B. Chem. Rev. 2012, 112, 5967−6026.
(18) (a) Fachel, F. N. S.; Schuh, R. S.; Veras, K. S.; Bassani, V. L.;
Koester, L. S.; Henriques, A. T.; Braganhol, E.; Teixeira, H. F.
Neurochem. Int. 2019, 122, 47−58. (b) Petersen, M.; Abdullah, Y.;
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c02855
(19) So, Y.; Lee, S. Y.; Han, A.-R.; Kim, J.-B.; Jeong, H. G.; Jin, C. H.
Molecules 2016, 21, 1083.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partly supported by JSPS KAKENHI grant
number JP17K08337.
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