Natural Products and Bioprospecting (2020) 10:51–56
https://doi.org/10.1007/s13659-020-00232-6
ORIGINAL ARTICLE
A New Epi‑neoverrucosane‑type Diterpenoid from the Liverwort
Pleurozia subinflata in Borneo
Takashi Kamada1,2 · Mary Lyn Johanis2 · Shean‑Yeaw Ng2 · Chin‑Soon Phan2 · Monica Suleiman2 ·
Charles S. Vairappan2
Received: 9 January 2020 / Accepted: 6 February 2020 / Published online: 15 February 2020
© The Author(s) 2020
Abstract
New bioactive 13-epi-neoverrucosane diterpenoid, 5β-acetoxy-13-epi-neoverrucosanic acid (1) along with three known
secondary metabolites, 13-epi-neoverrucosan-5β-ol (2), chelodane (3) and (E)-β-farnesene (4) were isolated from the MeOH
extract of east Malaysia’s liverwort Pleurozia subinflata. The chemical structure of new compound was elucidated by the
analyses of its spectroscopic data (FTIR, NMR and HR-ESI-MS). These epi-neoverrucosane-type compounds seem to be
notable chemosystematic markers for P. subinflata in Borneo. Compound 3 was widespread in marine sponges however this
is the first record for 3 to be found in liverwort. These metabolites were tested for their antifungal potentials against selected
fungi from the marine environment. Compound 1 exhibited effective antifungal activity against Lagenidium thermophilum.
Graphic Abstract
Keywords Epi-neoverrucosane · Diterpenoid · Pleurozia subinflata · Liverwort · Borneo
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13659-020-00232-6) contains
supplementary material, which is available to authorized users.
* Takashi Kamada
takashi.kamada800@gmail.com
1
Department of Materials and Life Science, Faculty
of Science and Technology, Shizuoka Institute
of Science and Technology, 2200-2 Toyosawa, Fukuroi,
Shizuoka 437-8555, Japan
2
Institute for Tropical Biology and Conservation, Universiti
Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia
1 Introduction
Liverworts are the largest group of pioneering land plants
which arose during the adaptation of plants from marine to
terrestrial environment [1]. They produced terpenoids and/
or aromatic compounds as their major lipophilic constituents [2–5]. Many types of sesquiterpenoids from liverworts
are the enantiomer to those metabolites from higher plants
[6]. While, diterpenoids such as clerodanes, dolabellanes,
fusicoccanes, kauranes, labdanes, pimaranes and others were
found in numerous liverworts [6]. Recently several bioactive
13
Vol.:(0123456789)
52
cyathane diterpenoids were discovered [7, 8]. Cyathane is
precursor structure for biosynthesis of the verrucosanetype diterpenoid, a fused 3,6,6,5-tetracyclic carbon skeleton [9]. First verrucosane diterpenoid was isolated from
Mylia verrucosa [10]. Later, some analogs such as neoverrucosane-, homoverrucosane-, epi-neoverrucosane- and
epi-homoverrucosane-type were reported [11–13]. The latest epi-neoverrucosane analog was reported in 2013 [14].
Hereby, we report yet another new epi-neoverrucosane diterpenoid, 5β-acetoxy-13-epi-neoverrucosanic acid (1) was
isolated, along with three known secondary metabolites,
13-epi-neoverrucosan-5β-ol (2), chelodane (3) and (E)-βfarnesene (4) from east Malaysia’s liverwort Pleurozia subinflata (Fig. 1).
Besides, liverworts have long been used as traditional
medicine by the indigenous people in some parts of China.
In the past half-century, Prof. Yoshinori Asakawa (Tokushima Bunri University, Japan) has begun to study the chemical composition of liverworts collected from Asia, Europe
and South America, many of which have reported to have
diverse chemical structures and exhibited numerous biological activities [2, 6]. Thus, research focusing on the biological
activity of liverwort and industrial use are significant. Our
research examined the antifungal effects of the four isolated compounds against selected fungi separated marine
organisms.
2 Results, Discussion and Conclusion
Compound 1 was isolated as colorless oil and analyzed
for the molecular formula C22H34O4 by HR-ESI-MS [M
− H] − ion at m/z 361.2391. The 13C and 1H NMR data
(Table 1) indicated the presence of an isopropyl unit at
δC 31.1, 23.2 and 22.3; δH 1.51–1.55, 1.00 and 0.78, one
carboxylic carbon at δC 183.0, an acetoxy unit at δC 171.5,
21.5; δH 2.04, one oxygenated methine at δC 75.1; δH 5.28,
two tertiary methyls at δC 25.5 and 16.4; δH 1.12 and 0.87,
Fig. 1 Chemical structures of
1–4
13
T. Kamada et al.
Table 1 1H and 13C NMR (600 and 150 MHz) data of 1 (in CDCl3, δ
in ppm, J in Hz).
No
δC
1
2
3
44.1
26.7
21.8
4
5
6
20.1
75.1
42.3
7
8
36.8
34.9
9
32.3
10
11
52.0
36.3
12
27.5
13
14
15
16
17
18
19
20
5-OAc
45.3
51.7
31.1
22.3
23.2
25.5
16.4
183.0
171.5
21.5
δH
2.20 (1H, dd, J = 4.3, 13.1)
0.65 (1H, ddd, J = 4.1, 4.1, 8.3)
0.69 (1H, dd, J = 4.3, 8.3)
0.56 (1H, dd, J = 4.3, 4.3)
5.28 (1H, dd, J = 7.6, 10.7)
1.76 (1H, dd, J = 7.6, 12.5)
0.84 (1H, m)
1.51–1.55 (1H, m)
1.09–1.11 (1H, m)
1.81–1.84 (1H, m)
1.59–1.62 (1H, m)
2.45 (1H, ddd, J = 1.4, 8.9, 11.6)
1.21–1.23 (1H, m)
1.81–1.84 (1H, m)
1.51–1.55 (1H, m)
1.88 (1H, m)
1.67 (1H, dd, J = 7.6, 13.1)
1.51–1.55 (1H, m)
0.78 (3H, d, J = 6.9)
1.00 (3H, d, J = 6.9)
1.12 (3H, s)
0.87 (3H, s)
2.04 (3H, s)
six methylenes, four methines, and three quaternary
carbons which corresponding well to HSQC spectrum.
Based on these findings, six degrees of unsaturation was
A New Epi-neoverrucosane-type Diterpenoid
calculated from HR-ESI-MS, and it attributed to two carbonyl units and one tetracyclic ring for 1.
The 1H–1H COSY experiment revealed the spin systems
as depicted by the bold lines in Fig. 2. In the HMBC spectrum, the three-bond correlations of H3–C(16) and H3–C(17)
to the opposite carbons C(17) and C(16), and to C(13) and
C(15), allowed the placement of isopropyl unit at C(13)
which was further confirmed by 1H–1H COSY. The acetoxy
unit at C(5) was confirmed by HMBC correlations between
H–C(5) to 5-OAc. The downfield shifted of 13C and 1H
NMR at C(5) further supported this deduction. The HMBC
correlations of H2–C(11) to C(20); and H–C(14) to C(20)
suggested the carboxylic carbon at C(10). These findings
together with HMBC correlations of H3–C(18) to C(2), C(3),
C(4) and C(5); and H3–C(19) to C(1), C(6), C(7) and C(8)
permitted establishment for the planar structure of 1 (Fig. 2).
The relative stereochemistry of 1 was deduced from the
NOESY correlations (Fig. 2) and comparison of its chemical shift, coupling constants and NOE correlations with
those of known analogs [10–14]. The NOE correlations of
H–C(5) to H2–C(6α) (δH 1.76), H3–C(18) and H3–C(19);
and H–C(14) to H3–C(19) have suggested H–C(5), H–C(14),
H3–C(18) and H3–C(19) on α relative configuration. While,
NOE correlations of H–C(1) to H2–C(3β) (δH 0.56) and
H2–C(6β) (δH 0.84) showed H–C(1) must be on another
face, β relative configuration. The earlier NOE cross peak
of H–C(1) to H2–C(3) (δH 0.56) has led to the assignment
of H2–C(3) (δH 0.56) on β configuration, therefore H2–C(3)
(δH 0.69) must be on α configuration. With this finding, the
configuration at H–C(2) can be assigned based on vicinal
coupling constants of cyclopropane unit between H–C(2)
and H2–C(3α) (3J2-3α = 8.3 Hz) and between H–C(2) and
H2–C(3β) (3J2-3β = 4.3 Hz). These coupling constant values suggested H–C(2) has a cis relationship with H2–C(3α)
within the cyclopropane unit, therefore α configuration was
assigned for H–C(2). While, the carboxyl unit at C(10) was
assigned on β configuration due to a trans-fused at C/D ring
junction. Thus, the relative configurations of 1R*, 2S*, 4S*,
5R*, 7S*, 10S* and 14R* were determined as identical to
53
those of known analogs of neoverrucosane and epi-neoverrucosane [10–14]. To distinguish epi-neoverrucosane
from neoverrucosane, the NOE correlations of H–C(1) to
H–C(15) and H3–C(17); and H–C(13) to H–C(14), showed
13-isopropyl unit to H–C(14) has a trans configuration, suggested a epi-neoverrucosane. Furthermore, similar NOE correlations of H–C(1) to H–C(15); and H–C(13) to H–C(14)
were observed in 12-acetoxy-13-epi-neoverrucosann-5-one
[14]. On the contrary, these NOE were not observed in
neoverrucosane-type, neoverrucosan-5β,9β-diol, instead
H–C(13) to H–C(20) was detected [15]. Thus, the structure
1 was established without confusion. The configuration of
isopropyl unit at C(13) generated during formation of tricyclic system (Fig. 3) determined the biosynthesis of neoverrucosane or epi-neoverrucosane [9]. To the best of our
knowledge, compound 1 was considered as first discovery
of 13-epi-neoverrucosane that containing a carboxyl moiety
or even among related skeletons such as verrucosane and
neoverrucosane. The methyl at C(20) of 1 might have followed a three-step oxidation, via a hydroxyl and carbonyl,
to the corresponding carboxylic acid [16], as shown in the
purposed biosynthetic pathway (Fig. 3).
The known compounds were identified as 13-epineoverrucosan-5β-ol (2) [11], chelodane (3) [17], and (E)β-farnesene (4) [18], after compared its spectroscopic data
with published literatures. The tetracyclic diterpenes are
relatively rare in nature, and mainly found in the species of
Plagiochila, Jamesoniella and Fossombronia [6]. However,
we found epi-neoverrucosane-type diterpene derivatives (1
and 2) from east Malaysia’s liverwort, Pleurozia subinflata.
These secondary metabolites seem to be the good chemosystematic markers for P. subinflata in Borneo. Compound 3
was widespread in marine sponges such as Chelonaplysilla
erecta, Raspailia sp. and even in Sigmosceptrella sp. [17,
19, 20]. However, this is the first record for 3 found in liverwort. Compound 4 was the most common farnesane-type
sesquiterpene in liverworts. It was distributed throughout
more than 20 Jungermanniales and Pleuroziales species
including Pleurozia [6].
Fig. 2 1H–1H COSY, key
HMBC and NOE correlations
of 1
13
54
T. Kamada et al.
Fig. 3 Proposed biosynthetic pathway of 1
1 exhibited effective antifungal activity (MIC values of
12.5 μg/mL) against Lagenidium thermophilum.
Table 2 The MIC (μg/mL) of 1–4 against six fungal strains
Strains
MIC (µg/mL)
1
L. thermophilum
H. sabahensis
F. moniliforme
F. oxysporum
F. solani
O. humicola
2
12.5
50.0
> 100.0
> 100.0
> 100.0
> 100.0
3
100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
4
100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
Positive control: itraconazole MIC 2.0 µg mL−1
Compounds 1–4 were evaluated its biological potentials
against fungal strains isolated from the Bornean ocean,
Lagenidium thermophilum IPMB 1401, Haliphthoros
sabahensis IPMB 1402, Fusarium moniliforme NJM 8995,
Fusarium oxysporum NJM 0179, Fusarium solani NJM
8996 and Ochroconis humicola NJM 1503 (Table 2). The
minimum inhibition concentration (MIC) values of compound 1 against L. thermophilum and H. sabahensis were
12.5 and 50 μg/mL, respectively. While, compounds 2 and
3 showed MIC values of 100 μg/mL against L. thermophilum. Compound 4 was inactive (> 100 μg/mL) against the
tested fungi.
In conclusion, this is the first time ever since 2013 of the
last epi-neoverrucosane being discovered from nature. A
new 13-epi-neoverrucosane diterpenoid, 5β-acetoxy-13epi-neoverrucosanic acid (1) along with three known
secondary metabolites, 13-epi-neoverrucosan-5β-ol (2),
chelodane (3) and (E)-β-farnesene (4) were found in east
Malaysia’s liverwort Pleurozia subinflata. Compound
13
3 Experimental Section
3.1 General Experimental Procedures
Optical rotation was taken on the automatic polarimeter
(AUTOPOL IV automatic polarimeter) in chloroform solutions at 28 °C. IR spectrum was recorded on the FTIR
spectroscopy (Perkin Elmer). NMR spectra were recorded
on the 600 MHz FT-NMR (Jeol) using deuterated chloroform (CDCl3) with tetramethylsilane (TMS) as the internal standard. MS spectra were obtained using LC-ESIIT-TOF-MS (Shimadzu). For preparative TLC, Merck
Kieselgel 60 F254 was used and Kieselgel 60 was used for
column chromatography. Purification was performed using
high performance liquid chromatography (LC-10 AT, Shimadzu) equipped with UV detector.
3.2 Biological Materials
Specimens of P. subinflata (M. Suleiman & S.-Y. Ng 5946)
were collected from Mount Trus Madi (5° 33′ 13.1″ N,
116° 30′ 41.9″ E), Sabah, Malaysia in August 2015. The
specimens were identified based on external morphology
by the fifth author. A voucher specimen (BORHB0026)
is deposited in the BORNEENSIS Herbarium at Institute
for Tropical Biology and Conservation (ITBC), Univeristi
Malaysia Sabah (UMS).
A New Epi-neoverrucosane-type Diterpenoid
3.3 Extraction and Isolation
The air-dried liverwort specimens (42 g) were extracted
using 100% methanol (MeOH) (1.0 L × 3 each for two
days). The crude extract was partitioned between distilled
water (150 mL) and ethyl acetate (EtOAc) (50 mL × 3).
After removal of the organic solvent, the EtOAc fraction
(500 mg) was chromatographed on a Si gel column using
hexane (Hex) and EtOAc system as eluent with increasing
polarity (Hex/EtOAc: 9:1, 8:2, 7:3, 5:5, 100% EtOAc) to
yield five fractions, 1–5. Fraction 2 (76 mg) was subjected to
repeated preparative TLC with toluene to yield 2 (8.8 mg), 3
(7.4 mg) and 4 (15.4 mg). Fraction 3 (60 mg) was subjected
to repeated preparative TLC with hexane/EtOAc: 7:3, and
the resulted sub-fraction was further purified by semi-preparative high performance liquid chromatography (HPLC) to
yield 1 (12.8 mg). The isolation was operated using a reverse
phase C18 column (5 μm, 10 mm × 250 mm) measured at
UV wavelength of 210 nm under gradient elution with the
following conditions: 40–100% acetonitrile (MeCN)/H2O.
3.3.1 5β‑Acetoxy‑13‑Epi‑neoverrucosanic Acid (1)
Colorless oil; [α]28.0
D + 67.8 (c = 0.5, CHCl3); IR νmax 3488,
3060, 1735 and 1712 cm−1; 1H NMR (CDCl3, 600 MHz) δ
5.28 (1H, dd, J = 7.6, 10.7 Hz, H-5), 2.45 (1H, ddd, J = 1.4,
8.9, 11.6 Hz, H-11α), 2.20 (1H, dd, J = 4.3, 13.1 Hz,
H-1), 2.04 (3H, s, OAc), 1.88 (1H, m, H-13), 1.81–1.84
(1H, m, H-12α), 1.81–1.84 (1H, m, H-9α), 1.76 (1H, dd,
J = 7.6, 12.5 Hz, H-6α), 1.67 (1H, dd, J = 7.6, 13.1 Hz,
H-14), 1.59–1.62 (1H, m, H-9β), 1.51–1.55 (1H, m, H-8α),
1.51–1.55 (1H, m, H-12β), 1.51–1.55 (1H, m, H-15),
1.21–1.23 (1H, m, H-11β), 1.12 (3H, s, H-18), 1.09–1.11
(1H, m, H-8β), 1.00 (3H, d, J = 6.9 Hz, H-17), 0.87 (3H, s,
H-19), 0.84 (1H, m, H-6β), 0.78 (3H, d, J = 6.9 Hz, H-16),
0.69 (1H, dd, J = 4.3, 8.3 Hz, H-3α), 0.65 (1H, ddd, J = 4.1,
4.1, 8.3, H-2), 0.56 (1H, dd, J = 4.3, 4.3 Hz, H-3β); 13C
NMR (CDCl3, 150 MHz) δ 183.0 (C, C-20), 171.5 (C, OAc),
75.1 (CH, C-5), 52.0 (C, C-10), 51.7 (CH, C-14), 45.3 (CH,
C-13), 44.1 (CH, C-1), 42.3 (CH2, C-6), 36.8 (C, C-7), 36.3
(CH2, C-11), 34.9 (CH2, C-8), 32.3 (CH2, C-9), 31.1 (CH,
C-15), 27.5 (CH2, C-12), 26.7 (CH, C-2), 25.5 (CH3, C-18),
23.2 (CH3, C-17), 22.3 (CH3, C-16), 21.8 (CH2, C-3), 21.5
(CH3, OAc), 20.1 (C, C-4), 16.4 (CH3, C-19); negative ion
HRESIMS: m/z 361.2391 (calcd for C22H33O4 [M–H]−,
361.2384).
3.4 Antifungal Assay
The minimum inhibitory concentration (MIC) of the fungistatic on hyphae was performed by incorporating the pure
compound solutions (12.5, 25.0, 50.0, 100.0 μg/mL) onto
PYGS agar in a petri dish followed by inoculation of six
55
tested fungal strains [21–23]. The MIC was determined
visually as the lowest concentration showing no hyphal
growth after they were incubated at 25 °C for 7 days.
Acknowledgements This research was financially supported by the
Sabah Biodiversity Centre Grant (SaBC) [No. GL0070] and Universiti Malaysia Sabah (UMS) Grant [SBK0258-SG-2016]. The authors
would like to thank Prof. Dr. Kishio Hatai (Borneo Marine Research
Institute, UMS) for his kind guidance on bioassay. We are grateful
to the Sabah Forestry Department for their support and assistance in
the field. Finally, we were able to conduct research using literatures
purchased through research project A provided by Shizuoka Institute
of Science and Technology.
Funding This research was financially supported by SaBC Grant
[No. GL0070] and UMS Grant [SBK0258-SG-2016].
Compliance with Ethical Standards
Conflict of interest The authors declare no conflict of interest.
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References
1. L. Hui, R.X. Xu, X.S. Zhang, T.T. Zhu, H.X. Lou, A.X. Cheng,
Phytochemistry 159, 190–198 (2019)
2. Y. Asakawa, A. Ludwiczuk, J. Nat. Prod. 81, 641–660 (2018)
3. S.Y. Ng, T. Kamada, C.S. Vairappan, Rec. Nat. Prod. 11, 508–
513 (2017)
4. S.Y. Ng, T. Kamada, M. Suleiman, C.S. Vairappan, Nat. Prod.
Res. 32, 1832–1837 (2018)
5. S.Y. Ng, T. Kamada, C.S. Phan, M. Suleiman, C.S. Vairappan,
Heterocycles 96, 1958–1965 (2018)
6. Y. Asakawa, A. Ludwiczuk, F. Nagashima, in Chemical Constituents of Bryophytes, ed. by A.D. Kinghorn. Progress in
the Chemistry of Organic Natural Products, vol 95 (Springer,
Vienna, 2013), p. 796
7. J. Wei, Y. Cheng, W.H. Guo, D.C. Wang, Q. Zhang, D. Li, J.
Rong, J.M. Gao, Sci. Rep. 7, 8883–8896 (2017)
8. J. Wei, W.H. Guo, C.Y. Cao, R.W. Kou, Y.Z. Xu, M. Górecki,
L.D. Bari, G. Pescitelli, J.M. Gao, Sci. Rep. 8, 2175–2189
(2018)
9. P.M. Dewick, Nat. Prod. Rep. 19, 181–222 (2002)
10. A. Matsuo, H. Nozaki, M. Nakayama, S. Hayashi, J. Chem. Soc.
Chem. Commun. 5, 198–200 (1978)
11. C. Grammes, G. Burkhardt, M. Veith, V. Hugh, H. Becker, Phytochemistry 44, 1495–1502 (1997)
12. H.J. Liu, C.L. Wu, H. Becker, J. Zapp, Phytochemistry 53,
845–849 (2000)
13
56
13. H. Shimogawa, T. Teruya, K. Suenaga, H. Kigoshi, Bull. Chem.
Soc. Jpn. 78, 1345–1347 (2005)
14. A.J. Singh, J.D. Dattelbaum, J.J. Field, Z. Smart, E.F. Woolly,
J.M. Barber, R. Heathcott, J.H. Miller, P.T. Northcote, Org. Biomol. Chem. 11, 8041–8051 (2013)
15. A. Spyere, D.C. Rowley, P.R. Jensen, W. Fenical, J. Nat. Prod.
66, 818–822 (2003)
16. A. Banerjee, B. Hamberger, Phytochem. Rev. 17, 81–111 (2018)
17. A. Rudi, Y. Kashman, J. Nat. Prod. 55, 1408–1414 (1992)
18. Y. Asakawa, M. Toyota, H. Tanaka, T. Hashimoto, D. Joulain,
J. Hattori Bot. Lab. 78, 183–188 (1995)
13
T. Kamada et al.
19. T. Yosief, A. Rudi, Z. Stein, I. Goldberg, G.M.D. Gravalos, M.
Schleyer, Y. Kashman, Tetrahedron Lett. 39, 3323–3326 (1998)
20. J.M. Ryan, Ph.D Dissertation, Victoria University of Wellington, 2007
21. T. Kamada, C.S. Phan, T. Hamada, K. Hatai, C.S. Vairappan, Nat.
Prod. Commun. 13, 17–19 (2018)
22. T. Kamada, J. Kulip, K. Tani, C.S. Phan, K. Hatai, C.S. Vairappan,
Rec. Nat. Prod. 12, 317–322 (2018)
23. K.J. Shamsudin, C.S. Phan, J. Kulip, K. Hatai, C.S. Vairappan, T.
Kamada, J. Asian Nat. Prod. Res. 21, 435–441 (2019)