K. Hostettmann

M. Neves

R. Morais

S. Gafner

English

Crossref

Three Triterpenoids and one Flavonoid from
the Liverwort Asterella blumeanaGrown In
Vitro
M. Neves1, R. Morais1*, S. Gafner2, K. Hostettmann2
1 Escola Superior de Biotecnologia- UCP, Rua Dr. Bernardino de Almeida, 4200 Porto, Portugal
2 Institut de Pharmacognosie et Phytochimie, Universite´ de Lausanne, B.E.P., CH-1015, Lausanne, Switzerland
This is the first report on the chemical constituents of Asterella blumeana. Three triterpenoids
(22-hydroxyhopane, 22,29-dihydroxyhopane, 6a,22-dihydroxyhopane) and one flavonoid (isoscutellarein-
7,8,4'-trimethyl ether) were isolated from the CH2Cl2 extract of the liverwort Asterella blumeanagrown
in vitro. The structures of isolated compounds were established by spectroscopic methods (UV, EI mass
spectroscopy,1H- and 13C-NMR). 22,29-dihydroxyhopane is a new natural compound.# 1998 John Wi-
ley & Sons, Ltd.
Phytother. Res.12, S21–S24 (1998)
Keywords:Liverworts, in vitro cultures,Asterella blumeana, triterpenoids, flavonoid.
INTRODUCTION
Liverworts are known to be a rich source of terpenoids
and phenolic compounds which constitute the oil bodies.
From these plants a high diversity of substances has been
isolated, including new structure types, some of them
with interesting biological activities (Asakawa, 1989).
Since only a few species of liverworts have been
phytochemically investigated (less than 5% of known
species), many interesting substances remain to be
isolated. However, advances on the study of the
chemistry of these plants are hindered by the difficulty
in collecting a sufficient quantity of pure material of
certain species. Their small size and the fact that
liverworts usually grow mixed with other bryophytes
and are strongly attached by their rhizoids to the soil,
make their purification a difficult and time consuming
task (Becker and Harrison, 1987; Asakawa, 1989).In
vitro cultures of liverworts can be a good alternative,
producing sufficient and homogenous amounts of plant
material for subsequent analysis (Oneet al., 1992; Takazi
et al., 1995).
The genusAsterellacontains approximately 80 species
(Frahm and Frey, 1983) but only a few have been studied
phytochemically:Asterella augusta, A. odora; A. aus-
tralis; A. tenera, A. lindenbergiana; A. venosa(Asakawa
and Heidelberger, 1982). In this work, we report the
isolation and structural elucidation of three hopane type
triterpenoids and one flavonoid, fromin vitro cultures of
Asterella blumeana.The structures are shown in Figures
1 and 2.
MATERIAL AND METHODS
Plant material and culture conditions. Plants of the
liverwort Asterella blumeana(Nees) Pande´ Srivastava et
Khen grownin vitro, obtained from the culture collection
of autotrophic organisms of the Institute of Botany,
Trebon, Czech Republic, were subcultured on fresh B5
medium (Gamborget al., 1968) every 2 months and
maintained in a photoperiod of 16 h light (30 mol/m2 s) at
22°C.
Plant material extraction. Air dried Asterella blumeana
plants (147 g) were ground and then extracted at room
temperature with CH2Cl2 to give 7.51 g of extract.
Isolation procedure.A portion of the CH2Cl2 extract of
Asterella blumeanaplants (7 g) was submitted to CC on
silica gel using mixtures of petrol, EtOAc and MeOH of
increasing polarity, giving frs 1–20. Fr 8 (400 mg) was
fractionated by CC on Sephadex LH-20 with MeOH-
CHCl3 (1:1), giving fractions A–E. Compound1
(100 mg) was isolated from frs C–D by crystallization
with CHCl3 and MeOH. Fraction 14 (500 mg) was
fractionated by CC on Sephadex LH-20 with MeOH-
CHCl3 (1:1) giving fractions A–F. Fr C (210 mg) was
further chromatographed over CC silica gel with petrol-
EtOAC (9:1) resulting in the isolation of compound2
(50 mg). Frs 15–16 were subjected to CC on Sephadex
PHYTOTHERAPY RESEARCH, VOL.12, S21–S24 (1998)
CCC 0951–418X/98/0S0S21–04 $17.50
# 1998 John Wiley & Sons, Ltd.
* Correspondence to: R. Morais, Escola Superior de Biotecnologia- UCP, Rua
Dr. Bernardino de Almeida, 4200 Porto, Portugal.
Contract/grant sponsor: JNICT; Contract/grant number: (BD/2848/93-IF).
Accepted 11 June 1997
LH-20 with MeOH-CHCl3 (1:1),to affordfractionsA–H.
Fr F (36mg) was further fractionatedon CC silica gel
with MeOH-CHCl3 (99:1)resultingin theisolationof the
compounds3 (29mg) and4 (2.6mg).
General. 1H and13C spectraweremeasuredin CDCl3 at
200.06and50.30MHz respectively.TMS: int. standard.
TLC silica gel 60 F254Al sheets(Merck). CC silica gel
(40–63mm, Merck, 600 55mm i.d., 250 21mm i.d.
and 115 15mm i.d.) 1H and 13C NMR: Varian VXR
200, EI- MS and D/CI-MS: Finnigan MAT TSQ-700
Triple stagequadrupoleinstrument.
RESULTS AND DISCUSSION
Dried plantsof Asterellablumeanagrown in vitro were
extractedwith CH2Cl2. The fractionationof the extract
by a combinationof silica gel columnchromatography,
gel filtration on SephadexLH-20 and recrystallization,
resultedin the isolationof threetriterpenoidscontaining
thehopaneskeleton(1–3) andoneflavonoid(4).
Compound 1
White crystals.On SiO2 TLC (EtOAc- petrol, 1:1) Rf
0.61 and on Diol TLC (hexane-isopropanol,95:1) Rf
0.28.ThiscompoundwasnotUV activeontheTLC plate
andacquiredaviolet colourationwith Godinreagent.MP
229.0°C–230.5°C. [a]D =39.6° (c = 1, CHCl3). EI-MS
andDCI-MSgavemolecularweightfor 1 of m/z= 428.In
13C-NMR andDEPT,30 carbonswerefound:11 CH2; 8
CH3; 5 CH and 6 quaternarycarbons(Table 1). With
these data, a molecular formula of C30H52O was
proposed.Finally, 1 was identified as the triterpenoid
22-hydroxyhopaneby comparisonof 1H- and13C-NMR
data with literature values(Mahato and Kundu, 1994;
Grammeset al., 1994;Agetaet al., 1993;Kamayaet al.,
1990).Tsudaet al. (1961)describedthe isolationof 22-
hydroxyhopanefrom the fern Diplopterygiumglaucum.
Grammeset al. (1994) describedthe isolation of this
compoundfrom the liverworts Fossombroniaalaskana
andF. pussilla.
Compound 2
White crystals.On SiO2 TLC (EtOAc- petrol, 1:1) Rf
0.45 and on Diol TLC (hexane-isopropanol,95:1) Rf
0.19.On TLC plateit wasnot UV activeandacquireda
pink colouration with Godin reagent. MP 193,7°C–
195.1°C. [a]D =60.3° (c = 1, CHCl3). EI-MS andDCI-
MS datagavea molecularweight for 2 of m/z= 444. In
13C-NMR andDEPTweredetected30carbons:10CH2,8
CH3,6 CH and6 quaternarycarbons(Table1). Thus,the
molecularformulaobtainedwasC30H52O2. The
1H- and
13C-NMR of this compoundwere very similar to the
spectraof 1, but in thecaseof 2, in 13C-NMR acarbonat
69.3ppm(correspondingto anoxygenatedmethine)was
detected.By comparisonwith literaturedata(Mahatoand
Kundu,1994;Wongetal., 1986;Elix etal., 1982),2 was
identified as zeorine(6a, 22-dihydroxyhopane).Elix et
al. (1982)reportedthe isolationof this compoundfrom
the lichen Heterodermiatremulansand Morais (1990)
described its isolation from the liverwort Reboulia
hemispheaerica.Although 2 did not presentbiological
activity againstCladosporiumcucumerinumandCandi-
da albicans (data not shown), Wong et al. (1986)
reportedcytotoxic activity toward culturedP-388cells
(ED = 1.1mg/mL).
Compound 3
White crystals.OnSiO2 TLC (EtOAc-petrol,1:1),3 had
Rf 0.34andon Diol TLC (hexane-isopropanol,95:1)Rf
0.11.On theTLC platewasnot UV activeandacquired
violet colouration with Godin reagent.MP 230.9°C–
232.7°C. [a]D =ÿ6.5° (c = 1, CHCl3). EI-MS andDCI-
Table 1. 13C-NMR chemicalshifts of compounds1, 2 and 3
Carbon 1a 2a 3b
1 40.3 40.4 40.5
2 18.6 18.5 19.0
3 42.1 43.8 42.3
4 33.2 33.6 33.4
5 56.1 61.1 56.3
6 18.6 69.4 19.0
7 33.2 45.5 33.5
8 41.8 42.9 42.1
9 50.3 49.5 50.7
10 37.4 39.4 37.6
11 20.9 21.1 213
12 24.1 24.0 24.5
13 49.8 49.8 50.1
14 41.9 41.9 42.1
15 34.4 34.4 34.6
16 21.9 21.9 22.8
17 53.9 54.0 54.4
18 44.1 44.0 44.5
19 41.2 41.2 419
20 26.6 26.6 25.8
21 51.1 51.1 47.0
22 73.9 73.9 75.0
23 33.4 36.7 33.5
24 21.6 22.1 21.8
25 16.1 17.1 16.0
26 15.8 18.3 16.9
27 17.02 17.1 17.2
28 16.7 16.1 16.2
29 28.7 28.8 70.1
30 30.8 30.9 25.5
a The values given are ppm from internal Me4Si in CDCl3
solution.
b C5D5N solution.
Table 2. Long-range correlations detected in the HMBC
spectrum of compound3
1H 13C
CH2-29 21, 22, 30
(1H 3.88 d, J=10.3 Hz
1H 3.85 d, J=10.3 Hz)
CH- 21 17, 20, 22, 29, 30
(1H 2.68 q, J=10 Hz)
CH3- 23 (3H 0.89 s) 3, 4, 5, 24
CH3- 24 (3H 0.83 s) 3, 4, 5, 23
CH3- 25 (3H 0.83 s) 1, 9, 10
CH3- 26 (3H 0.98 s) 8, 9, 14
CH3- 27 (3H 1.00 s) 7, 8, 14, 15
CH3- 28 (3H 1.02 s) 13, 17, 18, 19
S22 M. NEVESET AL.
# 1998JohnWiley & Sons,Ltd. Phytother.Res.12, S21–S24(1998)
MS gavea molecularweight for 3 of m/z= 444. In 13C-
NMR andDEPT,30carbonswerefound:7 CH3, 12CH2,
5 CH and 6 quaternarycarbons.With these data, a
molecular formula of C30H52O2 was proposed. An
oxygenatedmethylene at 70.1 ppm was detectedin
13C-NMR (Table1). As this compoundhadonemethyl
grouplessthan1 and2,althoughthespectraweresimilar,
it was possibleto concludethat a methyl group was
substitutedby a-CH2-OH.In orderto determinetheexact
position of the CH2-OH in molecule 3, HMBC and
HMQC experimentswerecarriedout. Finally, basedon
the long-rangecorrelationsdetectedin HMBC spectrum
(Table 2), the structureof 22,29-dihydroxyhopane was
established. Galbraith et al. (1964) described the
chemicalsynthesisof this compound,but to our knowl-
edgeit wasneverreportedto havebeenisolatedfrom a
naturalsource.
Compound 4
Yellow powder.Rf (SiO2 TLC EtOAc-petrol1:1)= 0.38.
Rf (Diol TLC hexane-isopropanol95:5)= 0.17. On the
TLC plate it was UV active at 254nm and acquireda
yellow colourationwith Godinreagent.EI-MS spectrum
gaveamolecularweightfor 4 of m/z= 328.In 13C-NMR
spectrumof 4 weredetected18carbons:3 CH3 (at61.65,
56.32and55.53correspondingto methoxygroups),6 CH
and 9 quaternary carbons. Its UV spectrum was
characteristicof an aromaticcompound.Threesinglets
at3.96ppm,3.95ppmand3.94ppmweredetectedin 1H-
NMR spectrumof 4 correspondingto three methoxy
groups. In the aromatic region of the spectrumtwo
signalsat 7.91ppm(2H, dd, J = 7 Hz, 2 Hz) andat 7.04
ppm (2H, dd, J = 7 Hz, 2 Hz) gave evidenceof the
presenceof a para substituted aromatic ring. Two
singletscorrespondingto two aromatichydrogenswere
alsopresentat 6.59ppm andat 6.43ppm (Table3). By
comparisonof 13C-NMR spectrumof 4 (Table 3) with
literaturedata,a greatsimilarity to thoseof salvigenin
(Ring B) and wigertin (ring A and C) was found
(Markham et al., 1982). The existenceof a methoxy
groupin position8, wasdeducedfrom theEI-MS data.In
thecaseof 8-OCH3 flavone,theintensityof thepeakM

ÿ15is higherthanpeakM (molecularion),whereasthis
is the oppositefor 6-OCH3 flavones(Goudardet al.,
1978,1979).Finally, this compoundwasidentifiedas5-
OH-7,8,4'-trimethoxyflavone (isoscutellarein-7,8,4'-tri-
methyl ether) by comparisonof its EI-MS, 1H-NMR
and UV spectra with literature data (Morais, 1990).
Isoscutellarein-7,8,41-trimethyl ether was first isolated
from Citrus reticulata (Iimuma et al., 1980). Morais
(1990)describedtheisolationof this compoundfrom the
liverwort Rebouliahemispheaerica.
This is the first reporton the chemicalconstituentsof
Asterellablumeanaand the first time that triterpenoids
have been isolated from this genus.22,29-dihydroxy-
hopaneis a newnaturalcompound.
Acknowledgement
M. Neveswould like to thankJNICT for thescholarshipgranted(BD/
2848/93-IF).
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Figure 2. Structure of compound 4.
TRITERPENOIDSFROM ASTERELLABLUMEANA S23
# 1998JohnWiley & Sons,Ltd. Phytother.Res.12, S21–S24(1998)
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S24 M. NEVESET AL.
# 1998JohnWiley & Sons,Ltd. Phytother.Res.12, S21–S24(1998)


Three triterpenoids and one flavonoid from the liverwort Asterella blumeana grown in vitro

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Three triterpenoids and one flavonoid from the liverwort Asterella blumeana grown in vitro

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