Cytotoxic, radical scavenging and antimicrobial activities of sesquiterpenoids from the Tahitian liverwort Mastigophora diclados (Brid.) Nees (Mastigophoraceae)

Article abstract of DOI:10.1007/s11418-010-0423-8

A drimane, (+)-drimenol (1), five known herbertanes, (-)-α- herbertenol (2), (-)-herbertenediol (3), mastigophorene A (4), (-)-mastigophorene C (5) and (-)-mastigophorene D (6), a pimarane, (-)-e nt-pimara-8(14),15-dien-19-oic acid (7), and two eudesmanolides, (-)-diplophyllolide A (8) and (-)-diplophyllin (9) were isolated from the Tahitian Mastigophora diclados (Brid.) Nees. Herbertane sesquiterpenes (2, 3, 5 and 6) showed cytotoxicity against HL-60 and KB cell lines, radical scavenging activity and antimicrobial activity against Bacillus subtilis. (-)-Diplophyllolide A (8) also exhibited cytotoxicity against HL-60 and KB cell lines.

Full text of DOI:10.1007/s11418-010-0423-8

J Nat Med (2010) 64:417–422
DOI 10.1007/s11418-010-0423-8
ORIGINAL PAPER
Cytotoxic, radical scavenging and antimicrobial activities
of sesquiterpenoids from the Tahitian liverwort Mastigophora
diclados (Brid.) Nees (Mastigophoraceae)
•
Ismiarni Komala Takuya Ito Fumihiro Nagashima
•
•
•
Yasuyuki Yagi Yoshinori Asakawa
Received: 10 February 2010 / Accepted: 29 March 2010 / Published online: 12 May 2010
Ó The Japanese Society of Pharmacognosy and Springer 2010
Abstract A drimane, (?)-drimenol (1), five known her-
bertanes, (-)-a-herbertenol (2), (-)-herbertenediol (3),
mastigophorene A (4), (-)-mastigophorene C (5) and (-)-
components have also been found in Herbertus species
[10–16], thus Mastigophora and Herbertus were concluded
to be closely related chemically [4]. Herbertane sesquit-
erpenoids are chemical markers of Mastigophora diclados
and it has been reported to possess interesting biological
activities. The dimeric herbertanes mastigophorene A (4),
B and D (6) have been found to exhibit neurotrophic
activity [6], while a-herbertenol (2), b-herbertenol, her-
bertenediol (3), herbertenal, 1,2-diacetoxyherbertene (10)
prepared from 3, mastigophorene C (5) and mastigopho-
rene D (6) inhibited NO production in lipopolysaccharide
(LPS)-stimulated RAW 264.7 cells [17]. (-)-a-Herbertenol
(2), (-)-b-herbertenol, (-)-a-formylherbertenol and (-)-
b-bromoherbertenol showed antifungal activity against
plant pathogenic fungi Botrytis cinerea and Rhizoctonia
solani [16]. a-Herbertenol (2), b-herbertenols, a-formyl-
herberternol and mastigophorene C (5) showed antimicro-
bial activity against bacterium Staphylococcus aureus [7].
During a search for biologically active metabolites of
liverworts, we found that Et2O and MeOH crude extracts of
the Tahitian Mastigophora diclados exhibited cytotoxicity
against HL-60 and KB cell lines, 1,1-diphenyl-2-picryl-
hydrazyl (DPPH) free radical scavenging activity and
antimicrobial activity against Staphylococcus aureus
NBRC 15035 and Bacillus subtilis NBRC 3134 as shown
in Table 1. Recently, we analysed the ether crude extract of
the Tahitian M. diclados using GC-MS and reported the
presence of herbertene (30.4%), a-herbertenol (2) (15.4%),
b-herbertenol (1.4%) and herbertenediol (3) (21.9%) [18].
Here we report the isolation, structural elucidation and
derivatization of chemical components of the Tahitian M.
diclados as well as results of their cytotoxic, radical
scavenging and antimicrobial activities. Structures were
established by a combination of extensive NMR, IR and
mass spectrometry. Cytotoxic, radical scavenging and
mastigophorene
D (6), a pimarane, (-)-ent-pimara-
8(14),15-dien-19-oic acid (7), and two eudesmanolides,
(-)-diplophyllolide A (8) and (-)-diplophyllin (9) were
isolated from the Tahitian Mastigophora diclados (Brid.)
Nees. Herbertane sesquiterpenes (2, 3, 5 and 6) showed
cytotoxicity against HL-60 and KB cell lines, radical
scavenging activity and antimicrobial activity against
Bacillus subtilis. (-)-Diplophyllolide A (8) also exhibited
cytotoxicity against HL-60 and KB cell lines.
Keywords Liverwort ꢀ Mastigophora diclados ꢀ
Sesquiterpenoid ꢀ Cytotoxicity ꢀ Radical scavenging ꢀ
Antimicrobial activity
Introduction
The liverwort Mastigophora diclados (Brid.) Nees has
been classified into the Mastigophoraceae family [1] and is
known to contain monomers and dimers of herbertane
sesquiterpenoids, macrocyclic bisbibenzyls, ent-trachylo-
bane and ent-pimarane diterpenoids [2–9]. These
I. Komala ꢀ T. Ito ꢀ F. Nagashima ꢀ Y. Yagi ꢀ Y. Asakawa (&)
Faculty of Pharmaceutical Sciences, Tokushima Bunri
University, Yamashiro-cho, Tokushima 770-8514, Japan
e-mail: asakawa@ph.bunri-u.ac.jp
I. Komala
Pharmacy Department, Faculty of Medicine and Health
Sciences, Syarif Hidayatullah State Islamic University,
Jl. Ir. H. Juanda No. 95 Ciputat, Jakarta 15412, Indonesia
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J Nat Med (2010) 64:417–422
Table 1 Cytotoxic, radical scavenging and antimicrobial activities of sesquiterpenoids from the Tahitian Mastigophora diclados
Compounds
Cytotoxicity IC50 (lg/ml)
Radical scavenging
IC50 (lg/ml)
Antimicrobial
MIC (lg/ml)
HL-60
KB
Et2O crude extract
2.4 0.5
14.6 0.8
21.4 0.7
20.8 0.7
B. subtilis (16)
S. aureus (16)
MeOH crude extract
13.1 2.8
32.6 1.5
B. subtilis (64)
S. aureus (64)
(?)-Drimenol (1)
–
–
–
–
(-)-a-Herbertenol (2)
(-)-Herbertenediol (3)
Mastigophorene A (4)
(-)-Mastigophorene C (5)
(-)-Mastigophorene D (6)
(-)-ent-Pimara-8(14),15-dien-19-oic acid (7)
(-)-Diplohyllolide A (8)
(-)-Diplophyllin (9)
12.8 0.7
12.5 0.6
–
B. subtilis (8)
1.4 0.1
11.8 1.4
1.9 0.6
–
B. subtilis (8)
–
–
–
2.4 0.1
14.8 1.1
2.7 0.8
2.0 0.1
[100
[100
–
B. subtilis (8)
2.5 0.4
14.2 1.0
B. subtilis (2)
[100
1.4 0.2
[100
3.3 1.2
[100
[100
–
–
–
(-)-1,2-Diacetoxyherbertene (10)
(-)-1-Hydroxy-2-methoxyherbertene (11)
Mytomicin C
2.6 0.0
15.0 0.5
[100
23.2 0.7
–
B. subtilis (16)
11.0 0.9
14.9 1.2
B. subtilis (32)
0.016 0.001
0.011 0.001
–
Vitamin C
–
–
–
–
–
–
2.6 1.0
1.9 0.6
–
–
Quercetin
–
Ampicillin
\ 2
–, Not tested because the isolated compounds were obtained in low yields (1–3 mg)
´
´
antimicrobial activities were evaluated by using WST-8
colorimetric, DPPH and dilution assays, respectively.
of the Universite de Polynesie Franc¸aise (UPF). The vou-
cher specimen was deposited in UPF and Tokushima Bunri
University.
Materials and methods
Extraction and isolation
General experimental procedures
The air-dried and ground Mastigophora diclados (106.5 g)
was extracted with Et2O and then MeOH. Filtration and
removal of solvent in vacuo gave the Et2O (3.5 g) and
MeOH (4.0 g) crude extracts, respectively. Fractionation of
Et2O extract (3.5 g) upon silica gel (hexane–EtOAc gra-
dient) gave eleven fractions. Fraction 5 (2.2 g) was further
purified (silica gel, Sephadex LH-20, preparative HPLC,
and hexane–EtOAc) to give (-)-a-herbertenol (2, 3 mg)
[a]D -62.9 (c 1.0, CHCl3) [lit. -55.0] [14], (-)-herber-
tenediol (3, 49 mg) [a]D -33.5 (c 1.0, CHCl3) [lit. -46.5]
Optical rotations were measured on JASCO P 1030 polar-
imeter. IR spectra were recorded on a Shimadzu FTIR-8400S
1
infrared spectrometer. The H- and 13C-NMR were mea-
sured on Varian unity-600 (1H, 600 MHz; 13C, 150 MHz)
and Varian Unity-500 MHz (1H, 500 MHz; 13C, 125 MHz)
instruments. Chemical shift values were expressed in d
(ppm) downfield from trimethylsilane (TMS) as an internal
standard, and in d 77.03 (ppm) from CDCl3 as a standard
(13C-NMR). Mass spectra were obtained on JEOL JMS 700
instrument. Column chromatography was performed on sil-
ica gel 60 (0.063–0.200 mm) (Merck) and Sephadex LH 20
(Amersham Pharmacia Biotech, eluant MeOH–CH2Cl2 1:1).
HPLC was carried out on Cosmosil 5SL-II (10 9 250 mm).
[15], mastigophorene
A (4, 1 mg), (-)-ent-pimara-
8(14),15-dien-19-oic acid (7, 15 mg) [a]D -155.3 (c 1.0,
CHCl3) [lit. -120.6] [19], (-)-diplophyllolide A (8, 5 mg)
[a]D -110.4 (c 1.0, CHCl3) [lit. -132.0] [20], (-)-diplo-
phyllin (9, 3 mg) [a]D -79.4 (c 1.0, CHCl3) [lit. -108.0]
[21] and (?)-drimenol (1, 2 mg) [a]D ?54.4 (c 1.0, CHCl3)
[lit. ?20.0] [22]. Fraction 6 (165 mg) was purified by using
a combination of column chromatography on silica gel
(hexane–EtOAc gradient), Sephadex LH-20 and pre-
parative HPLC (hexane–EtOAc 4:1) to give (-)-mastigo-
phorene C (5, 5 mg) [a]D -60.7 (c 1.0, CHCl3) [lit.
Plant material
Mastigophora diclados (Brid.) Nees was collected at
Mount Marau, 1400-m altitude (Tahiti) October 2007. The
species was identified by Dr. A. Pham from the Herbarium
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J Nat Med (2010) 64:417–422
419
-46.7]) [5], while purification of fraction 7 (231 mg) using
HPLC (hexane–EtOAc 3:2) gave (-)-mastigophorene D
(6, 17 mg) [a]D -68.2 (c 1.0, CHCl3), [lit. -46.1] [5].
Fractionation of MeOH extract (4.0 g) upon silica gel
(hexane–EtOAc gradient) gave ten fractions. Fraction 7
(100 mg) was purified upon silica gel, preparative HPLC
(hexane–EtOAc 3:2) and Sephadex LH-20 to give (-)-
herbertenediol (3, 54 mg) and (-)-diplophyllolide A (8,
2 mg). Fraction 8 (136 mg) was further purified (silica gel,
Sephadex LH-20, preparative HPLC, hexane–EtOAc gra-
dient and hexane–EtOAc 3:2) to give (-)-mastigophorene
D (6, 7 mg). The 1H-NMR and 13C-NMR spectra of
compounds 1–10 were identical to authentic samples.
pharyngeal squamous carcinoma), both purchased from DS
Pharma Biomedical, Osaka, Japan. HL-60 cells were non-
adherent cells and maintained in RPMI-1640 supplemented
with 10% fetal bovine serum (FBS) at 37°C in a humidified
atmosphere of 5% CO2. KB cells were grown as monolayer
in MEM supplemented with 10% FBS and 1% non-
essential amino acid at 37°C in a humidified atmosphere of
5% CO2. HL-60 or KB cells were seeded in 96-well plates
at a density 5 9 103 cells/well (100 ll/well), and the cul-
tures were incubated for 3 h (HL-60 cells) or 24 h (KB
cells). Then, 100-ll aliquots of medium containing test
compounds were added, and the cells were incubated for
72 h (HL-60 cells) or 48 h (KB cells). Cytotoxicity assays
were performed by adding 20 ll of WST-8 solution to the
cultures and incubating for 1–2 h at 37°C; after incubation,
the optical densities at 450 nm were measured by using a
microplate reader (Molecular Device, Tokyo, Japan).
Acetylation of herbertenediol (3)
(-)-Herbertenediol (3, 7 mg) was dissolved in a mixture of
Ac2O (1 ml) and pyridine (2 ml), and then the solution was
stored at room temperature overnight. The solution was
evaporated by using a freeze dryer and then extracted with
EtOAc. Purification upon silica gel (hexane–EtOAc 4:1)
gave (-)-1,2-diacetoxyherbertene (10, 9 mg) [a]D -51.2 (c
1.0, CHCl3) [lit. -22.6] [7, 16].
DPPH free radical scavenging assay
The pure compounds were spotted on TLC plates and then
the plate developed and dried. The TLC plate was sprayed
with 0.2% of 1,1-diphenyl-2-picrylhydrazyl (DPPH)
methanolic solution and stored in the dark. The spots on
TLC plates were observed under ordinary light after
30 min [23]. This method was used to observe whether
these compounds were active or not. In order to determine
the IC50 values of the active compounds, the scavenging
activity of pure compounds on 1,1-diphenyl-2-picrylhy-
drazyl (DPPH, Nacalai Tesque, Kyoto) was measured
according to a previous method [24]. Various concentra-
tions of samples (0.1 ml) were mixed with 0.1 ml of
50 lM DPPH solution in 96-well microtiter plates. After
30 min of incubation at room temperature the optical
densities at 517 nm were measured by using a microplate
reader (Molecular Device, Tokyo, Japan). The percentage
of scavenging of DPPH radical on samples was calculated
by using the following equation:
Methylation of herbertenediol (3)
(-)-Herbertenediol (3, 12 mg) was dissolved in acetone
(1 ml), and then K2CO3 (7 mg) and MeI (100 ll) were
added. The solution was refluxed for 1 h. Work-up as usual
gave (-)-1-hydroxy-2-methoxyherbertene (11, 1.6 mg) as
needle crystals, m.p. 36–37°C; [a]D -65.0 (c 1.0, CHCl3);
HR-EIMS: 248.1780, calcd. 248.1776 for C16H24O2; FTIR
(CHCl3) mmax 3525, 2956, 2871, 1595, 1487, 1460, 1417,
1371, 1292, 1230, 1166, 1145, 1058 cm-1 1H-NMR
;
(CDCl3, 500 MHz): 0.74 (3H, s, H-15), 1.17 (3H, s, H-14),
1.41 (3H, s, H-13), 1.53 (1H, m, H-10a), 1.64 (1H, s, H-
10b), 1.75 (1H, m, H-8a), 1.75 (2H, m, H-9), 2.28 (3H, s,
H-12), 2.60 (1H, m, H-8b), 3.86 (3H, s, OCH3), 5.84 (1H, s,
OH), 6.57 (1H, s, H-3), 6.72 (1H, s, H-5); 13C-NMR
(CDCl3, 125 MHz): 20.5 (C-9), 21.5 (C-12), 22.7 (C-13),
25.6 (C-14), 27.1 (C-15), 39.1 (C-8), 41.2 (C-10), 44.8 (C-
11), 51.2 (C-7), 56.1 (OCH3), 109.0 (C-3), 121.8 (C-5),
127.0 (C-4), 132.5 (C-6), 142.5 (C-1), 146.4 (C-2). EI-MS
m/z (rel. int.): 248 [M?] (88), 233 (4), 205 (5), 192 (12),
178 (64), 166 (100), 145 (13), 91 (14), 69 (7), 41 (9).
% inhibition ¼ 1 ꢁ [OD(DPPH + sample)/OD(DPPH)]
ꢂ 100%
Antimicrobial assay
The following microorganisms were used for testing anti-
microbial activity: Bacillus subtilis NBRC 3134 and Kleb-
siella pneunomiae NBRC 3512. Bacteria strains were tested
by using microdilution assays and minimum inhibitory
concentration (MIC) values were determined. Bacterial
strains were inoculated on YP agar plates (1% polypeptone,
0.2% yeast extract, 0.1% MgSO4ꢀ7H2O and 6% agar) and
were incubated at 37°C (B. subtilis) and 30°C (K. pneuno-
miae) for 12 h. The stock solution of crude extracts was
Biological assays
Cytotoxicity assay
Cell lines used in this study were HL-60 cells (human
promyelocytic leukaemia) and KB cells (human
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J Nat Med (2010) 64:417–422
prepared in 10 mg/ml in DMSO and further diluted to
varying concentration in 96-well plates which have con-
tained incubated microbial strains. Each plate was further
incubated in 37°C for overnight and ampicillin in varying
concentration was used as reference drug.
(Lepidoziaceae) [27], Targionia (Targioniaceae) [30],
Frullania (Frullaniaceae) [31] and Porella (Porellaceae)
[29].
Herbertane sesquiterpenoids (2–6) are chemical markers
of M. diclados and they have been found as major compo-
nents in the Tahitian M. diclados. Together with (?)-
drimenol and (-)-herbertanes, two (-)-eudesmanolides,
(-)-diplophyllolide A (8) and (-)-diplophyllin (9), were
also isolated from this liverwort. Previously, 8 and 9 have
been isolated from the liverworts Diplophyllum (Scapania-
ceae)[21],Plagiochila(Plagiochilaceae)[32],Clasmatocolea
(Lophocoleaceae) [33], Chiloscyphus (Lophocoleaceae) [33]
and Tritomaria (Scapaniaceae) [34].
Result and discussions
Mastigophora diclados was extracted with Et2O and
MeOH and then fractionated by using a combination of
column chromatography on silica gel, Sephadex LH-20 and
preparative HPLC to give (?)-drimenol (1) [22], (-)-a-
herbertenol (2) [14], (-)-herbertenediol (3) [6], mastigo-
phorene A (4), (-)-mastigophorene C (5), (-)-mastigo-
phorene D (6) [5, 6], (-)-ent-pimara-8(14),15-dien-19-oic
acid (7) [25], (-)-diplophyllolide A (8) [26] and (-)-dip-
lophyllin (9) [21] (Fig. 1). Acetylation and methylation of
(-)-herbertenediol (3) were carried out in order to compare
the biological activity of the resulting derivatives with the
natural herbertanes.
The isolated herbertanes (2, 3, 5 and 6) exhibited
cytotoxicity against HL-60 and KB cell lines, DPPH radi-
cal scavenging activity and antimicrobial activity against
Bacillus subtilis as shown in Table 1. Since these com-
pounds possess hydroxyl groups, the derivatization of the
major component (-)-herbertenediol (3) was carried out to
investigate whether the biological activities were caused by
the presence of hydroxyl groups or not. The herbertane
derivatives (-)-1,2-diacetoxyherbertene (10) and (-)-1-
hydroxy-2-methoxyherbertene (11) were tested for their
cytotoxic, radical scavenging and antimicrobial activities.
When tested against KB cells, there was no significant
decrease in cytotoxicity of 10 and 11 (relative to 3), sug-
gesting that the hydroxyl group does not play an important
role in providing cytotoxicity against KB cells. When they
were tested against HL-60 cells, 1,2-diacetoxyherbertene
(10) did not show any significant decrease in activity;
however, natural a-herbertenol (2) and 1-hydroxy-2-meth-
oxyherbertene (11) had reduced cytotoxic activity against
HL-60 cells as shown in Table 1. There is a possibility that
the position of substitution on the benzene ring will influ-
ence the cytotoxicity against HL-60 cells; however,
because of the lack of related compounds available for
testing, we cannot conclude which structural features
influenced the decrease in cytotoxicity of both compounds
(2 and 11) against HL-60 cells. (-)-Diplophyllolide A (8)
exhibited strong inhibitory activity against HL-60 and KB
cell lines with IC50 values of 1.4 and 3.3 lg/ml, respec-
tively. (-)-Diplophyllolide A (8) was reported to be toxic
against P388 cells [33], while (-)-diplophyllin (9) was
toxic against KB cells [21]. Ohta et al. [21] concluded that
the cytotoxicity of eudesmanolide is due to the presence of
the methylene lactone function. Among all the isolated
sesquiterpenes and derivatives tested, (-)-herbertenediol
(3) and (-)-diplophyllolide A (8) were the most inhibitory
active sesquiterpenes against the HL-60 cell line; on the
other hand, only (-)-diplophyllolide A (8) showed strong
cytotoxic activity against the KB cell line.
This is the first reported isolation of (?)-drimenol (1)
from a natural source, although it has been reported as a
synthetic compound [22]. The IR, MS and NMR spectra of
(?)-drimenol (1) were identical to those (-)-drimenol [27],
except for the sign of optical rotation. (-)-Drimenol has
been found in higher plants [28, 29] and the different liv-
erworts, Diplophyllum (Scapaniaceae) [21], Bazzania
Since the herbertanes (2, 3, 5 and 6) possess hydroxyl
groups, there was no doubt that the radical scavenging
Fig. 1 Isolated sesqui- and diterpenoids from the Tahitian Masti-
gophora diclados
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J Nat Med (2010) 64:417–422
421
8. Hashimoto T, Irita H, Takaoka S, Tanaka M, Asakawa Y (2000)
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functional group. This phenomenon was supported by the
negative result for (-)-1,2-diacetoxyherbertene (10) and
reduced activity of (-)-1-hydroxy-2-methoxyherbertene
(11) (relative to 3, 5 and 6) in the DPPH free radical
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The herbertane sesquiterpenoids (2, 3, 5 and 6) were
also tested for antimicrobial activity against Bacillus sub-
tilis and Klebsiella pneunomiae. These herbertanes were
active against B. subtilis as shown in Table 1, but only
herbertenediol (3) showed weak activity against K. pneu-
nomiae (MIC at 100 lg/ml). Among all of the herbertanes,
the dimeric herbertane (-)-mastigophorene D (6) is the
most active sesquiterpene against B. subtilis (MIC at 2 lg/
ml). Derivatization of herbertenediol (3) caused decreased
antimicrobial activity against B. subtilis, relative to (-)-
1,2-diacetoxyherbertene (10) and (-)-1-hydroxy-2-meth-
oxyherbertene (11) (Table 1).
16. Matsuo A, Yuki S, Nakayama M (1986) Structure of ent-her-
bertane sesquiterpenoids displaying antifungal properties from
the liverwort Herberta adunca. J Chem Soc Perkin Trans I 701–
710
17. Harinantenaina L, Quang DN, Nishizawa T, Hashimoto T,
Kohchi C, Soma G, Asakawa Y (2007) Bioactive compounds
from liverworts: inhibition of lipopolysaccharide-induced induc-
ible NOS mRNA in RAW 264.7 cells by herbertenoids and cu-
parenoids. Phytomedicine 14:486–491
18. Ludwiczuk A, Komala I, Pham A, Bianchini JP, Rahariveloma-
nana P, Asakawa Y (2009) Volatile components from selected
Tahitian liverworts. Nat Prod Commun 4:1387–1392
19. Shibata S, Mihashi S, Tanaka O (1967) The occurrence of (-
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Acknowledgments We thank Prof. L. Raharivelomanana and Prof.
´
´
Jean-Pierre Bianchini from Universite de Polynesie Franc¸aise (UPF)
for their collection of liverwort. Thanks are also due to Dr. A. Pham
(UPF) for his identification of the samples and Dr. A. Ludwiczuk for
her valuable discussion. I. Komala acknowledges JBIC loan/IP-530
for fellowship program development of Faculty of Medicine and
Health Sciences Syarif Hidayatullah State Islamic University (UIN)
Indonesia. This work was supported in part by Grant-in-Aid for the
Scientific Research (A) (No 11309012) from the Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan.
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