FLAVOUR AND FRAGRANCE JOURNAL, VOL. 1.

43-46 (1985)

An Approach to the Isolation of the Fragrant Principle of the

Liverwort Lophocolea heterophylla
Vlastimil Herout
Institute of Organic Chemistry & Biochemistry, Czechoslovak Academy of Science, Flemingovo ndm2sti 2, 166IO Prague 6,
Czechoslovakia. CSSR

The presence of a very fragrant component, C12H,,0, in the liverwort Lophocolea heterophyllu was proven. A
possible method for the isolation of this still unknown substance is discussed. The special ‘mossy’ note of this
natural product should be interesting as a possible component of fragrancies.

KEY WORDS Liverwort Lophocoleu heterophyllu C,,H,,O New class of aroma

INTRODUCTION detailed information on the very rich chemistry of

The study of odoriferous components of natural
fragrancies is a very old practice. Natural com-
ponents originating prevalently, but not exclu-
sively, from the plant kingdom were intensively
isolated from the beginning of the development of
modem chemistry. Today a rich variety of these
compounds is known, many of them being used in
large quantities in different branches of industry,
mainly for perfumes, cosmetics, etc. Furthermore,
many of the natural substances obtained from
essential oils or plant material extracts, are not
used directly, but only after some chemical mod-
ification. In a series of cases, the natural products
served as model structures for developing fully
synthetic preparations. The occurrence, methods
for isolation and identification, structure elucida-
tion, and the ways of their possible utilization
have been described in many textbooks (for
recent reviews of such data see, e.g., Herout,’
Boelens’ or Glasby3). Nevertheless, the fact
remains that some groups of fragrant compounds
were not easily accessible before the discovery of
the modem analytical and especially physico-
chemical equipment, which are now common in
laboratories. This is valid for some groups of
plants which, owing to their relatively small
growth, have never been accessible in the large
quantities necessary for classical isolation
methods. Such a situation was typical for the class
of liverworts (Hepaticae) representing a part of
the Bryophyta-their chemistry has been studied
only intensively during the last two decades,
partly in our Laboratories. For some more
0882-5734/85/0 10043-0450 1 .OO
@ 1985 by John Wiley & Sons, Ltd.
liverworts, see recent reviews of Markham and
P ~ r t e rHuneck’
,~ and of Asakawa.6 They contain,
mostly in their ‘oil bodies’ (typical oil droplets in
their cells) a plethora of components of very dif-
ferent classes, some peculiar to liverworts, others
are compounds encountered as secondary
metabolites commonly in higher plants (conifers
and angiosperms). The typical ‘mossy’ smell of
liverworts are due to terpenoids. This is remark-
able in that ‘true’ mosses do not practically con-
tain these compounds and are characterized by
the presence of triterpenoids, as the only iso-
prenoids. These terpenoid derivatives peculiar to
liverworts, exhibit mostly an inconspicuous smell.
Isolated in a purer state, they have a cedar wood
note. One well known exception is the presence
of bomyl acetate in some liverworts (see
Asakawa6), which has a characteristic ‘forest’
fragrancy like the needle essential oils of common
conifers, such as pine or spruce. According to our
experience, we have come across a special case
where the smell of a liverwort was highly original
and represented a very agreeable, unusually frag-
rant ‘mossy’ note. We published this fact some
time ago in a communication dealing with the
composition of components of the essential oil or
extracts of Lophocolea heterophylla (Schrad.)
Dum. (Jungermaniales)’.
Received 2 I December I984
44 V. HEROUT
In that paper, the main stress was given to
the presence of sequiterpenoids and further
waxy components, the non-fragrant ent-iso-
alantolactone [I] being the major component
of the plant extracts. The yield of the essential oil
prepared from this plantlet through distillation
with overheated steam, was very low, owing to
the low volatility of this sesquite enoid lactone.
In that essential oil we identified’p sesquiterpene
hydrocarbons, which are also found in other
related liverworts. In the ‘oxygen-containing vol-
atile fraction’, we were able to find a highly frag-
rant component with a probable formula
C,,H,,,O. This compound manifests its presence
very characteristically in any state, and it is poss-
ible to smell it after any mechanical contact with
the plantlet L. heterophylla; its tiny growth can
be seen in Figure 1. Any crushing of the cells of
that liverwort gives immediately a rise to the
characteristic odour. And it is useful to stress that
the content of this fragrant component does not
exceed several thousandths of one percent of the
dry weight of the plant specimen.
In the present paper I intend to call attention to
the existence of such a compound, since according
to my opinion, the intensity and agreeable note of
the odour of this liverwort compound could have
some practical use for perfumery. In further
experiments since the publication of the foremen-
tioned paper,’ we have tried repeatedly to establ-
ish the chemical nature of this fragrant com-
1 cm
I
Fig. 1. A tiny growth of a specimen of Lophocolea
heterophylla on a tree-bark
pound, but we failed to prepare any reasonable
amount of this rare natural product. There may
be several reasons for such a failure: (i) Even to
collect a larger amount of this liverwort is already
a difficult task. It is true that in the wet forests of
Czechoslovakia it grows quite commonly on
decaying logs or stumps of different trees, but
never in large quantities. The optimum possibility
to collect it, is to cut dense tufts, growing in com-
petition with mosses, green algae and filamentous
fungi, with a knife from horizontally-cut top of
the stumps. The portions of the plant growing on
the rough bark are only separated tediously from
this material. The purity of such specimens
remains, of course, doubtful. Nevertheless, in a
series of specimens collected at different times on
different trees in forests throughout a large part
of our country, we always found pratically the
same main components in the essential oil pre-
pared from these specimens. (ii) The stability of
the product which we obtained using the
preparative-scale GLC, always in very limited
amounts, was not high. In pure state it readily
polymerized. (iii) Last, but not least-failure may
be due to the situation when the NMR-instrument
is not well-tuned just at the time when you have
isolated (after a tedious procedure) a tiny amount
of a not fully stable product!
Since my personal program has left this area I
wish to publish our experience in the isolation and
structural elucidation of this compound to stimu-
late some well-equipped research workers (with
personal patience and laboratory instrumenta-
tion) to repeat and improve the methods used in
our Laboratory. It could be profitable if they were
interested in blending new fragrancies. The pers-
pective is such that nobody will be able to prepare
larger amounts of this fragrancy from natural
resources. But its structure-as it can be judged
from the relatively very simple molecular formula
C,,H,,,O-might be easy to synthetize with
reasonably good yields. Only then would it be
possible to gain this special fragrant natural com-
pound which could give to perfumes an until now
unexpected but certainly wanted note.
EXPERIMENTAL
Isolation of the Fragrant Principle
The mode of essential oil preparation and the
chromatographic separation into fractions con-
taining hydrocarbons and/or fragrant oxygen-
 FRAGRANT PRINCIPLE OF LOPHOCOLEA 45

207.AP-L
CHROMOSORB W
2’5m x4mm
170’ C

10
17
15 14
1
n I 1
t
18
c

Fig. 2. A typical run of the fragrant fraction of L. hererophylla. 1,2 solvents (CCI, + CHCI,); 3-fungal smell; 4-nitrogen-
containing compounds (unidentified); 5-not registered; 6-phenolic; 7-floral; 8-not registered; 9-pinene-like; 10-other
terpenoid; 1 1-oxygenated terpenoid; 12-not registered; 13-fresh note, weak; 14-very faint smell; 15-pine-needle smell;
1&not registered; 17-very intensive mossy fragrancy, clearly the main odoriferous component!; 18-very faint odour;
19-not registered; 20-spicy note; 21-faint note; 22-not registered; 23-lemon-like odour

containing compounds was published in sufficient it as the typical fragrant compount of L.

details elsewhere;’ the currently used analytical
and preparative methods are, also summarized in
a recent article (Walradts). In this paper, only the
GLC-analysis of the ‘oxygenated fragrant frac-
tion’ will be presented in detail. It was done with a
double evaluation, i.e., using splitted vent (1: 100)
(i) to flame ionization detector (FID)system of
F‘ye series 104 Chromatograph, Model 24, and
(ii) to an ‘odour port’, allowing simultaneous
evaluation of the aroma characters of single peaks
along with responses of FID record. In Figure 2 a
typical run is given: 16 peaks of very different
intensity were distinguished as smells by an
‘expert nose’ from about 23 peaks, also of a very
different amount, registered by FID.
Peak No 17 did not represent more than 5% of
the injected fraction (i.e., 3.0 mg of the fragrant
fraction), but its odour was of such an intensity,
that it was possible to smell its vapour in the
whole room (about 42 m3 volume) and recognize
heterophylla by all four persons present.
Comments to the Structure of the Fragrant Sub-
stance
Our newer experiments have not added any
further information in comparison with the data
published earlier’. Repeated mass spectroscopic
measurement just confirmed the molecular for-
mula, C12H200.
RESULTS
As a summary of our findings during the pro-
longed studies of the fragrant principle of L.
heterophylla I should like to point only to the fol-
lowing views: the unique character of this incon-
spicuous plant is its odour with a mossy note. For
me it is quite difficult to categorize this fragrance
46 V. HEROUT
into any of the now proposed classes of odours
(cf. e.g., AmooreY) without the possibility to
compare it with the reference compounds. It
belongs very probably to the class of cam-
phoraceous odours. Furthermore, the odour
intensity of this new compound is amazing. No
exact threshold value has been measured. But the
fact that ca 0.05-0.08 mg of the pure compound
eluted from the chromatograph into the atmos-
phere and diluted in the air (approx. 40 m3) was
identified without hesitation indicates its
threshold value to be very low. It seems to be
comparable with such intense odours as 5 a-
androsten-16-en-3-one (for its threshold value
see, e.g.9.
A11 these properties mentioned before allow
me to give a recommendation to follow this inter-
esting fragrance more in detail, to establish its
structure and to synthetize this compound quite
inaccessible in other ways. Giving such an advice
on the occasion of starting a new scientific journal
devoted to flavours and fragrance, I intend to give
a positive stimulus for all those who are interested
in finding new original perfume components.
REFERENCES
1 . V. Herout, in Fragrance Chemistry, E.T. Theimer ed.,
p. 221, Academic Press, New York (1982).
2. H. Boelens, ibid., p. 123.
3. J. S. Glasby, Encyclopediu ofrhe Terpenoids, Vol. I and 11.
Wiley, Chichester (1982).
4. K. R. Markham and L. J. Porter, in Progress in
Phyrochemisfry, J. B. Harborne and T. Swain, eds., Vol. 5,
p. 181. Pergamon Press, Oxford (1978).
5. S. Huneck, in New Manual of Bryology, R. M. Schuster
ed., Vol. 1, p. 1, Hattori Bot. Laboratory, Nichinan-shi
(Jap.) (1983).
6. Y. Asakawa, in Progress in the Chemistry of Organic
Nuturul Products, W.Herz, H. Greisbach and G. W. Kirby,
eds.. Vol. 42, p. 1, Springer-Verlag Wien, New York
(1982).
7. K. Konefnf, K. Ubik, S. Vasifkovh, M. Streibl and V.
Herout, CON. Czech. Chem. Commun., 47, 3164 (1982).
8. J. P. Walradt, Frugrunce Chemistry, E. T. Theimer ed.,
p. 525, Academic Press, New York (1982).
9. J. E. Amoore, ibid., p. 27.