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Review Article
Received: 15 January 2009, Revised: 6 February 2009, Accepted: 6 February 2009, Published online 11 March 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI 10.1002/ffj.1923
Flavour Fragr. J. 2009, 24, 105–116 Copyright © 2009 John Wiley & Sons, Ltd.
105
John Wiley & Sons, Ltd.
Lichen extracts as raw materials
in perfumery. Part 2: treemoss
Lichen extracts as raw materials in perfumery. Part 2: treemoss
Daniel Joulaina* and Raphaël Tabacchib
ABSTRACT: This is a comprehensive review of extracts from the lichen Pseudevernia furfuracea (treemoss) that are used in the
fragrance industry. Qualitative and quantitative analytical aspects are critically reviewed and the results are compared to
those of the related oakmoss extracts. It is shown that more than 90 constituents have been identified so far in treemoss
extracts, including 42 depsides, depsidones or depside-derived compounds, and 42 triterpenes or steroids. Constituents of
certain host trees, mainly Pinus species, generate specific analytical and toxicological issues which need to be considered in
addition to those related to the known degradation products of lichen compounds. A new classification of lichen extracts
used as raw materials in fragrance compounding is proposed. Copyright © 2009 John Wiley & Sons, Ltd.
Keywords: Pseudevernia furfuracea; treemoss; fragrances; depsides; triterpenes; steroids; resin acids; contact allergy
Introduction
Following on from the previous article on oakmoss (Evernia
prunastri),[1] the present review deals with treemoss extracts
that are used in fragrance compounding. In principle, so-called
treemoss extracts are mostly manufactured from Pseudevernia
furfuracea (L.) Zopf., a lichen which is particularly common on
coniferous trees, mainly pine, and cedar trees. Four chemical
races of P. furfura c e a have been defined: one contains mainly
physodic acid, a second physodic and olivetoric acids, a third
olivetoric acid and the last one physodic, olivetoric and oxyphysodic
acids (chemical structures are shown below).[2]
Lichens other than Evernia prunastri (true oakmoss) are also
processed industrially, such as Ramalina fraxinea (L.) Ach., some-
times admixed with Usnea caucasica Vain. and harvested mainly
from beech trees (Fagus spp.) in Macedonia.[3] When these
lichens are processed in the presence of E. prunastri, whatever
the proportions, the resinoids obtained should not be called
‘oakmoss’ but rather ‘treemoss resinoids’.
The Chemical Abstracts Service (CAS) has assigned various
Registry Numbers (RN) to treemoss extracts:
• 90028-67-4, defined as: ‘extractives and their physically
modified derivatives, etc. of Evernia furfuracea, Usneaceae’.
However, it should be pointed out that the correct botanical
and family names are, respectively, Pseudevernia furfuracea
and Parmeliaceae.
• 92129-88-9, defined as: ‘tree moss wax, Pseudovernia furfuracea.
Extractives and their physically modified derivatives Evernia
furfuracea’. Although this assignment is somewhat obscure,
one can consider that it corresponds to the same type of
products defined under RN 90028-67-4.
• 94944-93-1, defined as: ‘Evernia furfuracea extracts, ethanolysed’.
The same remark applies regarding the botanical name.
As mentioned previously,[1] this RN is likely to correspond to
most of the industrially available extracts, provided they are
produced from pure Pseudevernia furfuracea lichen, free of
exogenous products.
• 68648-41-9 and 68917-40-8, both defined as: ‘Extractives and
their physically modified derivatives, Evernia furfuracea and
Usnea barbata, Usneaceae’. Because another name under RN
68648-41-9 is ‘cedarmoss oil’, one can admit that the declared
lichens species grow specifically on cedar trees.
Considering this confusing situation, however, one can reasonably
admit that:
• CAS RN 94944-93-1 should be used specifically for absolute
oils prepared from concrete oils manufactured from pure
P. fur fura cea , whatever the host tree.
• CAS RN 68648-41-9 applies to any extractive (concrete or
absolute oils) of mixed lichen species, containing mainly
P. furfur ac ea and U. barbata and growing on cedar trees. As
a result, this RN corresponds to resin acid-less treemoss
extracts (see below).
• CAS RN 68917-40-8 applies to any other extract, whatever the
purity, origin, host tree, etc.
Therefore, extractives of impure P. f u r f u r a cea growing on pine
trees can be assigned this RN. Clearly, no RN has been assigned
to any extract obtained from Ramalina spp. in either pure form
or lichen mixtures.
As of 1997, about 1900 tons of such treemoss and 700 tons
of oakmoss were processed each year in France. In 2007, the
total quantity of treemoss and oakmoss processed by French
producers was only 540 and 550 tons, respectively (source:
Prodarom, Grasse, 9 January 2009).
Other related lichens are used for the production of industrial
fragrant extracts, such as Parmelia nepalensis, Usnea spp. and
Ramalina spp. in Nepal. However, this production did not exceed
ca. 60 tons in 1994.
* Correspondence to: D. Joulain, 15 Traverse de la Coste d’Or Supérieure,
F-06130 Grasse, France. E-mail: dajoulain@wanadoo.fr or daniel.joulain@
robertet.fr
aRobertet S.A., B.P. 52 100, F-06 131 Grasse cedex, France
bInstitut de Chimie, Avenue de Bellevaux 51, CH-2009 Neuchâtel, Switzerland
D. Joulain and R. Tabacchi
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As we pointed out in the first article,[1] the fragrance industry
indiscriminately indicated under the name of ‘oakmoss’ the
lichen collected on oak trees or the lichen collected on other
trees. However, distinction has been made traditionally between
‘cedarmoss’ collected mainly in Morocco on cedar trees (Cedrus
atlantica), and ‘treemoss’ gathered from pine trees (Pinus spp.),
although the lichen species is actually the same (P. fur fura c e a ).
The difference in fragrance is mainly attributable to the wood,
twigs, bark, etc. from pine trees, which contaminate the lichen
to various extents.[4]
In using similar solvents and standard procedures, and starting
from lichen containing 15–20% humidity, the extraction yields
are 5.5–6.0% and 2.5–3.0% for ‘pine treemoss’ and ‘cedar treemoss’,
respectively.
Several sources mention that extracts of Lobaria pulmonaria
(L.) Hoffm.—a foliose lichen, whereas E. prunastri and P. f ur fura ce a
are fruticose lichens—have been used in fragrance compounds.
Although the reality and importance of this remains unclear, this
lichen species is not processed industrially today.
Qualitative Composition of Treemoss Extracts
The chemical composition of treemoss (P. f u r f u r acea ) has been
thoroughly investigated, starting from the lichen itself and typical
industrial extracts.[5–14] It was reported that the lichen growing
on pine trees (Pinus sylvestris) is largely P. fu rf ur a cea, with minor
proportions of other species: Hypogymnia physodes, Usnea spp.,
Alectoria capillaries and Parmelia sulcata.[3,7] This generated mainly
qualitative data, which are reported in Huneck and Yoshimura’s
compilation.[25] As in the case of true oakmoss,[1] whatever the
extraction solvent or process, either batch or continuous,[24] the
duration and intensity of the hydrolytic pre-treatment can indeed
generate a variety of extracts with different compositional charac-
teristics.[3,23] This variability is drastically increased depending on
the origin of the lichen (see Introduction) and the host conifer tree,
viz. Pinus or Cedrus spp. When standard treemoss is collected on
pine trees in the Massif Central, France (Figure 1a), it is usually
made of 40–70% by weight of components (wood, bark, twigs,
needles, etc.) harvested from the tree itself. In some cases, this
proportion can even reach 80% (Figure 1b).
Apart from these analytical studies, which were focused on
the raw material that is used in the fragrance industry, a number
of papers have been published on lichen substances identified
in P. fur fura c e a . Classical analytical methodologies, including
separation and spectroscopic techniques for structure determi-
nation, are similar to those involved in similar cases.[1] Mono-aryl
compounds (Figure 2) are listed in Table 1, while depsides and
depsidones (Figure 4) are listed in Table 2.
Depsides are readily hydrolysed, and the intermediate
benzoic acid derivatives can be thermally decarboxylated. For
example, in the case of chloroatranorin, increasing the intensity
of the hydrolytic process (tepid or hot water or steam) results in
its almost complete degradation, with the production of methyl
β
-orcinol carboxylate 9, chloroatranol 15 and chlorohaematommic
acid 16.[3,23]
Direct analysis by gas chromatography–mass spectrometry
(GC–MS) of an extract of P. furfur a c ea growing on cedar trees
shows the presence of large amounts of olivetonide 20 and 5-(2-
oxoheptyl)-resorcinol 23, the latter not described,[21] and which
is likely to be an artifact generated during the analysis by ther-
mal decarboxylation of olivetonic acid 22 (Figure 3).[23]
Isopropyl haematommate 14 is clearly an artifact that is
formed from atranorin during the extraction with isopropanol.[14]
Rhizonic acid 19, already known from lichens, has been tenta-
tively identified in P. fu r fu ra ce a collected in Turkey, together with
a lactone which has been assigned the structure S1 (Figure 5).[26]
However, this structure might be corrected to that of vulpinic
acid 43, since the mass spectrum and the carbon nuclear mag-
netic resonance (CNMR) data are very similar. The chlorinated
amino-depside S2 claimed by the same authors is also unlikely,
at least as far as the B-ring of this depside is concerned,[26] which
should motivate further studies (e.g. hydrolysis experiments,
crystallization, melting point, X-ray analysis). Using an authentic
sample, perlatolic acid 31 was unambiguously identified by high
performance liquid chromatography–ion trap mass spectrometry
(HPLC–ITMS) operating in MS3.[13] Although it was found for the
first time in this lichen species, it had been identified previously
in other Parmelia spp.[22] Apart from atranorin 25 and chloroatra-
norin 26, which represent up to 80% of the depside fraction,[8]
olivetonide 20, physodone 32 and isophysodic acid 34 (which
Figure 1. (a) Pseudevernia furfuracea on pine tree; (b) raw ‘pine treemoss’ before processing.
Lichen extracts as raw materials in perfumery. Part 2: treemoss
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107
results from the rapid and total isomerization of physodic acid
35) are considered to be characteristic elements of industrial
extracts of P. furfur a c ea.[10,23] 2′-O-methylphysodone 33 originates
from the decarboxylation of the corresponding acid 36, previ-
ously recorded as a constituent of this species.[18]
It is worth mentioning the possibilities offered by the direct
analysis of a neat lichen sample by tandem mass spectrometry
(MS/MS), in placing it directly in the ionization chamber of
the mass spectrometer.[12] This technique bypasses the tedious
extraction and separations steps, which involve relatively large
amounts of lichen. In most cases, in addition to compiled data
on thin layer chromatography (TLC), negative chemical ionization–
MS/MS (NCI–MS/MS) provides decisive information for identifying
lichen substances, provided that a comprehensive database has
been assembled beforehand. As a result, one would expect
HPLC–MS/MS methodologies, which have recently been developed
Figure 2. Chemical structures of mono-aryl compounds identified in P. f ur f ur ac ea
Tab le 1. Monoaryl compounds identified in P. fu rf ura ce a extracts
Compound no. Name CAS RN MW References
12-Chloro-3,5-dimethoxytoluene NA 186.6 14
2Divarinol 500-49-2 151.2 14
35-Pentylresorcinol (= olivetol) 500-66-3 180.2 14,21
4Olivetolcarboxylic acid 491-72-5 224.2 21
5Orcinol 504-15-4 124.1 21
6Orcinol monomethylether 3209-13-0 138.1 21
7
β
-Orcinol 488-87-9 138.1 21
8
β
-Orcinolcarboxylic acid 4707-46-4 182.2 7,13
9Methyl
β
-orcinolcarboxylate 4707-47-5 196.2 7,15
10 Atranol 526-37-4 152.1 13
11 Haematommic acid 479-25-4 196.1 7,13
12 Methyl haematommate 34874-90-3 210.1 13
13 Ethyl haematommate 39503-14-5 224.2 13,14,35
14 Isopropyl haematommate NA 238.2 14
15 Chloroatranol 57074-21-2 186.6 6,13
16 Chlorohaematommic acid 56410-39-0 230.7 21
17 Ethyl chlorohaematommate 57857-81-5 258.7 13,35
18 Methyl orsellinate 3187-58-4 182.1 21
19 Rhizonic acid 479-26-5 196.2 26
20 Olivetonide 3734-54-1 248.3 7
21 Hydroxy-olivetonide NA 264.3 7
D. Joulain and R. Tabacchi
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for other families of natural products, to apply efficiently to lichen
substances as well.
Similarly, volatile compounds present in fresh lichens (P.
furfuracea and Evernia prunastri) can be analysed directly by
solid phase micro-extraction (SPME) sampling and analogous
sorptive trapping, followed by GC–MS. In this way, atranol and
chloroatranol (and other mono-aryl compounds) can be readily
detected in the volatile fraction from the crude lichen. This demon-
strates that hydrolysis and decarboxylation of atranorin and
chlorotranorin occurs in the 'living' lichen (ca. 15–20% humidity)
in the absence of any artificial treatment.[21] It is known that
depside hydrolases, present in unactivated form in the lichen,
can be activated again in the presence of water (rain water, etc.)
Furfuric acid 41 has been isolated in small amounts (2% of the
depside–depsidone fraction) by extraction of 2.4 kg P. f ur f urace a
from Massif Central (France) after careful cleaning from other
lichen species and elimination of wood and bark residues. The
structure of this unique depsidone has been confirmed by
synthesis[27] and also, in one step, by the acid-catalysed alkylation
of methyl
β
-orcinolcarboxylate 9 or atranorin 25, with physodalic
acid 39.[28] This reaction is so facile that it supports the claim that
41 is an artifact of the isolation procedure of P. f u r f u r a cea con-
taminated with Hypogymnia physodes.[28] However, subsequent
experiments confirmed the natural origin of 41, at least in this
specific chemotype of P. furfur ac ea. It appeared that the extrac-
tion conditions (hexane) were not sufficiently acidic to catalyse
the reaction. Indeed, when a mixture of Evernia prunastri (con-
taining methyl
β
-orsellinate and atranorin) and 10% of H. phys-
odes was extracted under the same conditions, only physodalic
acid was observed and isolated, whereas furfuric acid 41 was not
detected (R. Tabacchi, unpublished information). However, this
does not rule out a possible ‘in vivo relationship’ between the
two species, resulting in some enzyme-mediated reaction between
their metabolites.
In addition to the usual depsides and their derivatives present
in Parmelia species, the main constituent of commercial ‘tree-
moss’ resinoid obtained from Parmelia nepalensis (vide supra) is
protolichesterinic acid 24.[21] Not surprisingly, since this resinoid
is stated to be obtained by extraction with ethanol, it also
contains significant amounts of sugars (xylitol, arabitol, etc.)
which are easily identified by GC–MS after silylation.[21] Such
sugars have been previously identified in P. fu rfur ac ea.[29]
Starting from lichen collected on conifers (mainly P. sylvestris),
and after tedious separation of the lichen from any component
derived from the host tree, a number of sterols and triterpenes
have been identified by GC, UV, IR, NMR and MS.[11,30] They are
listed in Table 4, and their structures are shown in Figure 6. It is
highly probable that these compounds are metabolites from the
host tree (Pinus spp.),[13] involving a migration from the host to the
parasitic lichen. This hypothesis is well supported by two facts:
Tab le 2. Depsides and depsidones identified in Pseudevernia furfuracea extracts from various origins
Compound no. Name CAS RN MW References
25 Atranorin 479-20-9 374.3 5,6
26 Chloroatranorin 479-16-3 408.8 6,8
27 Lecanoric acid 480-56-8 318.3 19,20
28 Imbricaric acid 491-57-6 402.4 17
29 Microphyllinic acid 491-46-3 528.6 17
30 Olivetoric acid 491-47-4 472.5 8
31 Perlatolic acid 529-47-5 444.5 13,21
32 Physodone 58005-58-6 440.5 7,15
33 2′-O-methylphysodone 62806-12-6 454.5 7
34 Isophysodic acid 188347-29-7 470.5 7
35 Physodic acid 84-24-2 470.5 5,7,18
36 2′-O-methylphysodic acid 56484-74-3 484.5 7,18
37 3-Hydroxyphysodic acid 53899-46-0 486.5 7,16,18
38 Virensic acid 668-14-4 358.3 16
39 Physodalic acid 90689-60-4 416.3 8
40 Alectoronic acid 54226-87-8 510.6 18
41 Furfuric acid 100508-93-8 552.5 9
42 Fumarprotocetraric acid 489-50-9 456.3 17
Figure 3. Miscellaneous compounds identified in P. fu rf ura ce a. Mass spectrum
of 23 [m/z, (%)], 222 (M+, 35), 166 (3), 124 (23), 123 (50), 99 (100), 71 (62), 55 (29),
43 (100), 41 (32)
Lichen extracts as raw materials in perfumery. Part 2: treemoss
Flavour Fragr. J. 2009, 24, 105–116 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/ffj
109
1. Most of the sterols and triterpenoids listed in Table 4 have
been previously identified in Pinaceae,[31] such as Pinus monti-
cola,[32] P. lu ch ue ns is,[33] Picea jezoensis var. hodoensis.[34]
2. In a typical experiment, a 1 kg sample of ‘pine treemoss’ was
separated into three fractions: one part (A, 36%) was wood
devoid of lichen, a second part (B, 29%) comprised mainly
lichen (21%) intimately mixed with small pieces of wood, bark,
twigs etc. (8%), and a third ‘intermediate’ part (C, 35%) consisted
of large pieces of wood (22%) covered with some lichen (13%).
Each fraction was extracted using exactly the same procedure
and the concentration of
β
-sitosterol and stigmasterol (together),
chosen as target sterols, was measured by various methods,
including HPLC (Table 3).[13] The results confirm that these
sterols are components of the host tree and are also present
in the thallus of the lichen. A similar phenomenon is
observed with resin acids, which are well-known components
of Pinus (colophony, see below).
All this suggests that terpenoids and sterols biosynthesized in
the host tree migrate to the parasitic lichen.
It is well known that lichens can biosynthetize triterpenes and
steroids, even when growing in the absence of a host organism,
e.g. on rocks. The extent and truly parasitic nature of the rela-
tionship between the lichen and its host tree needs to be further
investigated with additional lichen species, and host trees that
are not conifers. However, it is now clearly established, at least in
the case of P. fur furacea growing on Pinus spp., that di- and triter-
penes, as well as steroids, biosynthesized in the host tree do
Figure 4. Chemical structures of depsides and depsidones identified in P. fu r fu ra ce a
D. Joulain and R. Tabacchi
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indeed migrate into the lichen. In the case of resinoids obtained
from lichen growing on cedar trees, one should expect to detect
terpenoids which are produced by the host tree (C. atlantica)
and which are also present in the well-known essential oil.
However, such studies are currently in progress and have so far
not been reported (D. Joulain, unpublished information).
Quantitative Composition of Treemoss Extracts
The content of defined lichen compounds in P. f ur fur ace a has only
rarely been measured. In most cases, the preferred analytical
technique is HPLC.[2,16] For example, atranorin 25, physodic 35,
hydroxyphysodic 37 and virensic acid 38 are claimed to represent
0.11–0.19%, 1.46–3.78%, 1.69–3.44% and 1.14–1.46%, respec-
tively, in dried lichen from Slovakia.[16] For the same reasons that
have been discussed previously in the case of oakmoss,[1] quanti-
tative data on the composition of industrial resinoids and other
extracts of P. furfur a c e a are even more scarce. For example, a
commercial treemoss absolute oil was found to contain 8.7%
methyl
β
-orcinolcarboxylate 9, 0.4% ethyl haematommate 13,
0.3% ethyl chlorohaematommate 17 and 0.3% 25.[35]
Contrary to depsides, which can undergo various degradations
(hydrolysis, decarboxylation, alcoholysis) to mono-aryl com-
pounds, which therefore may be readily available as reference
compounds, depsidones (viz. 35–42), present in larger quanti-
ties in treemoss than in oakmoss, do not generate such com-
pounds, but rather diphenyl ethers (32–34). Such ethers can be
quite diverse, depending on the extraction conditions, thus
drastically complicating the analytical task. Moreover, they are
not volatile and their contribution to the odour of the extracts
is negligible. However, nowadays, thanks to the availability of
efficient columns and instrumentation, GC–MS can be quite
effective for the analysis of mono-aryl compounds and even some
depsides, since silylated compounds with molecular weights
(MW) up to 800g/M can be eluted below 300 °C. For example,
persilylated atranorin (MW 590) is eluted at 230 °C on a 30 m
long non-polar column. A typical composition of an industrial
treemoss absolute oil is shown in Table 5.[13]
A global quantification method of polyphenols, using the
Folin–Ciocalteu method and 96-well plate assays, can be
applied with success to the rapid screening of many samples of
P. furfura ce a .[36]
The Resin Acid Issue in Commercial ‘Moss’ Resinoids
As we mentioned earlier[1] and have discussed again above, tree-
moss resinoids that are produced from the lichen collected from
pine trees contain variable amounts of resin acids (colophony),
depending on the proportion of 'wood' components. Colophony
is a complex mixture of diterpene acids (resin acids, ca. 90%),
including abietic acid 85 as the main constituent.[37] It has been
found that, when highly purified, abietic acid is non-allergenic.
However, it rapidly autoxidizes to the hydroperoxide 86, which is
identified as a major allergen in colophony.[38] Other oxidized
species derived from dehydroabietic acid (DHA) 87, such as 15-
hydroperoxy-dehydroabietic acid 88 and 7-oxodehydroabietic
acid (7-ODHA) 89, are also sensitizers (Figure 7). In particular, a
haptenation mechanism involving 89 has been proposed, which
may account for the allergic contact dermatitis (ACD) observed
from exposure to resin acids.[39] The qualitative monitoring of the
oxidation of diterpenoid resins can be achieved by HPLC coupled
with atmospheric pressure chemical ionization MS (LC–APCI–MS)
or APCI–MS/MS.[40]
In 2000, it was reported that products marketed under the
name ‘oakmoss absolute’ by two patch-tests suppliers, viz. Trolab
and Chemotechnique, actually contained resin acids.[41] This meant
that these products were mixtures of extracts, including one
obtained from P. fur fu race a growing on pine trees. As a conse-
quence, it was surmised that the use over the years of these
products in patch-testing for ACD to oakmoss may have been a
source of misdiagnosis. However, it was shown subsequently
that the diagnostic value of oakmoss absolute as an indicator
of fragrance ACD has been, and is, unaffected by the resin acid
contamination.[42] Concomitant observations showed that a strongly
statistically significant association between oakmoss absolute
and colophony (resin acids) triggers only a small increase in rates
of allergic response to colophony in oakmoss-positive patients.[43]
Figure 5. Unconfirmed structures of P. fu r fu ra ce a metabolites
Tab le 3. Distribution of diterpenes and selected sterols in
treemoss fractions[13]
Fraction of biomass (see text) A B C
Lichen content (%, w/w) 0 72 37
Sterols (%, w/w)a10.0 1.9 4.6
Total resin acid (%, w/w)b16.0 2.2 4.8
aAs
β
-sitosterol + stigmasterol, by HPLC. bBy GC–MS with silyla-
tion with internal standard.
Tab le 3. Distribution of diterpenes and selected sterols in
treemoss fractions[13]
Fraction of biomass (see text) A B C
Lichen content (%, w/w) 0 72 37
Sterols (%, w/w)a10.0 1.9 4.6
Total resin acid (%, w/w)b16.0 2.2 4.8
aAs
β
-sitosterol + stigmasterol, by HPLC. bBy GC–MS with silyla-
tion with internal standard.
Lichen extracts as raw materials in perfumery. Part 2: treemoss
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111
In July 2001, the International Fragrance Association (IFRA)
issued a recommendation (‘standard’) on oakmoss, stating:
‘In the presence of treemoss extracts, the level of oakmoss has to be
reduced accordingly such that the total amount of both extracts does
not exceed 0.1% in the final product. Oakmoss extracts used in perfume
compounds must not contain added treemoss. Treemoss contains resin
acids. The presence of resin acids can be detected by using a routine ana-
lytical method available from IFRA. However, traces of resin acids are
unavoidable in current commercial qualities of oak moss. As an interim
standard, these traces must not exceed 0.1% (1000 ppm) dehydroabietic
acid (DHA)'.
Figure 6. Serrattene derivatives identified in P. f ur fu ra ce a growing on pine trees
D. Joulain and R. Tabacchi
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A method for quantifying resin acids involves the determina-
tion of dehydroabietic acid (DHA) by HPLC with fluorimetric
detection, whilst assuming that DHA represents 40% of the total
resin acids.[44] This IFRA-recommended method (see below) is
efficient in every respect, and shows that typical commercial
treemoss absolute oils contain 5–6% w/w DHA. It is also sensi-
tive enough to detect and quantify DHA in a low ppm range
(<100 ppm) in contaminated oakmoss extracts. An alternative
global method for determining resin acids involves their sepa-
ration by gel permeation chromatography (GPC), followed by
methylation of the acids by diazomethane.[41] Using this method,
the contents of resin acids in two commercial samples of tree-
moss absolute oils were found to be 11.4% and 8.1%. In this
mixture, the concentrations of 7-oxo-dehydroabietic acid (7-
ODHA) were found to be 1.6% and 1.1%, respectively, but the
method of quantification of this single compound by gas
chromatography–mass spectrometry (GC–MS) is unknown.[41] In
contrast, the selective quantification of DHA and 7-ODHA can
be achieved by GC–MS with internal standardization.[13] It
requires two steps: the extraction of the acid fraction; followed
by in situ methylation with diazomethane and the trideuterated
esters of DHA (90) and 7-ODHA (91) as internal standards (IS).
Due to the excellent selectivity and sensitivity of this method,
quantification of DHA and 7-ODHA at the sub-ppm level is
possible in lichen absolute oils, and even in finished cosmetic
products.
Figure 6. (Continued)
Figure 7. Resin acids and their oxidation products (selection)
Lichen extracts as raw materials in perfumery. Part 2: treemoss
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113
A method has been developed for the selective removal of
resin acids from treemoss extracts, when manufactured from
lichen growing on pine trees.[45] Table 5 shows a list of concen-
trations of selected relevant constituents of treemoss before and
after such processing.[13] It is noteworthy that, although it still
contains the same normal levels of atranol and chloroatranol
but undetectable amounts of the atranorins, the resin acid-
depleted oil did not induce any reaction in two separate human
repeated insult patch tests (HRIPTs), according to the Marzulli–
Maibach protocol, on a total of ca. 200 volunteers at 3% concen-
tration (Robertet, unpublished data).[13] As we have demonstrated
above, resin acids are not only present in the wood debris from
the host pine tree, but they also migrate into the lichen. Since
dehydroabietic itself is not a sensitizer, it would be worthwhile
to monitor the oxidation rate of this hydrocarbon during its
migration in the thallus of the lichen. Would depsides, which are
polyphenols, act as anti-oxidants? In order to rule out a possible
bias or misinterpretation, the following experiment was carried
out: pure intact lichen (free of any wood debris) was soaked
briefly in hexane (3 min at 20 °C), yielding extract No. 1. Then,
the drained lichen was ground and extracted again with hexane
in a Soxhlet apparatus for 5 h, yielding extract No. 2. Quantification
of 7-ODHA was performed on extracts Nos 1 and 2 by GC–MS
with deuterated ISs, as above. The results demonstrate that it
is indeed present mainly in the thallus of the lichen (ca. 10 ppm),
whereas ca. 0.7 ppm only is recovered from its outer part.[13]
Tab le 4. Steroids and triterpenoids identified in Pseudevernia furfuracea
Compound no. Name CAS RN MW References
44 Cholestan-3
β
-ol 80-97-7 388.7 11,30
45 Cholest-5-en-3
β
-ol 57-88-5 386.6 11,30
46 Cholest-8-en-3
β
-ol 7199-91-9 386.6 11,30
47 Cholest-4-en-3-one 601-57-0 384.6 11,30
48 Ergostan-3
β
-ol 6538-02-9 402.7 11,30
49 Ergost-5-en-3
β
-ol 290299-12-6 400.7 11,30
50 Ergosta-5,22-dien-3
β
-ol (=brassicasterol) 474-67-9 398.6 11,30
51 Ergost-7-en-3
β
-ol (=fungisterol) 507-78-9 400.7 5,11,30
52 Ergosta-7,22-dien-3
β
-ol 17608-76-3 398.6 11,30
53 Ergosta-7,24(28)-dien-3
β
-ol 17105-77-0 398.6 11,30
54 Ergosta-5,8,22-trien-3
β
-ol (=lichesterol) 50657-31-3 396.6 11,30
55 Ergosta-5,7,22-trien-3
β
-ol (=ergosterol) 57-87-4 396.6 5,11,30
56 Ergosta-8,22-dien-3
β
-ol 36904-77-5 398.6 11,30
57 Ergosta-5,7,9(11),22-tetraen-3
β
-ol 26596-35-0 394.6 11,30
58 Ergost-4-en-3-one 51014-22-3 398.6 11,30
59 Stigmastan-3
β
-ol 83-45-4 416.7 11,30
60 Stigmast-5-en-3
β
-ol (=
β
-sitosterol) 5779-62-4 414.7 11,30
61 stigmast-4-en-3-one 1058-61-3 412.7 11,30
62 Stigmast-7-en-3
β
-ol (=schottenol) 6869-99-4 414.7 11,30
63 Stigmast-5,22-dien-3
β
-ol (=stigmasterol) 83-48-7 412.7 11,30
64 Stigmast-7,22-dien-3
β
-ol 18070-03-6 412.7 11,30
65 Ergosterol-5
α
,8
α
-peroxide 2061-64-5 428.6 11,30
66 3
β
-Hydroxy-5,8-epi-dioxyergosta-6,9(11),24(28)-triene 78342-37-7 426.6 11,30
67 Lanosterol 79-63-0 426.7 11
68 Lanost-9(11)-en-3
β
-ol (T ) 28032-52-2 426.7 11
69 24-Methyllanost-9(11)-en-3
β
-ol (T) NA 440.7 11
70 Lanost-9(11)-en-3
β
,24,25-triol NA 460.7 11
71 3
β
-Methoxylanost-9(11)-en-24,25-diol NA 474.7 11
72 24,25-Dihydroxylanost-9(11)-en-3-one 385384-28-1 458.7 11
73 4
α
-Methylergosta-7-24(28)-dien-3
β
-ol NA 412.7 11
74 4
α
-Methylergost-7-en-3
β
-ol 77122-68-0 414.7 11
75 Serrat-14-en-3
β
,21
α
-diol (=serratenediol) 2239-24-9 442.7 11
76 3
β
-Methoxyserrat-14-en-21
α
-ol NA 456.7 11
77 21
α
-Methoxyserrat-14-en-3
β
-ol NA 456.7 11
78 3
β
-Hydroxyserrat-14-en-21-one 3787-73-3 440.7 11
79 3
β
-Methoxyserrat-14-en-21-one NA 454.7 11
80 21
α
-Hydroxyserrat-14-en-3-one NA 440.7 11
81 Serrat-14-en-3,21-dione 1449-07-6 438.7 11
82 3
β
,21
α
-Dimethoxyserrat-14-en-24-ol NA 486.8 11
83 3
β
-Methoxyserrat-14-en-21
α
-30-diol 94805-72-8 472.7 11
84 3
β
-Methoxyserrat-14-en-21
α
-30-diol, diacetate NA 556.8 11
T, tentative identification; NA, no assignment.
D. Joulain and R. Tabacchi
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114
Moreover, the ratio of concentrations of 7-ODHA vs. DHA is higher
in the lichen than in the wood bearing the lichens. This suggests
that lichen compounds do not protect DHA against oxidation
and may even act as pro-oxidants. These results do not contradict
recent observations showing that methanol extracts of P. fu r fu ra ce a
have low antioxidant activity, in comparison with other lichen
species.[46]
Other Toxicological Issues
Whatever the type of lichen that is used for the manufacture of
treemoss resinoids, the corresponding absolute oils are expected
to contain significant levels of atranol 10 and chlorotranol 15,
since atranorin 25 and chloroatranorin 26 are major constitu-
ents of these lichens (Table 5). The latest opinion of the Scientific
Committee on Consumer Products (SCCP) concerning 10 and 15
is clearly focused on a use level based on elicitation (previously
sensitized individuals).[47] This new paradigm, if it becomes offi-
cial, overturns the present strategy of the safe use levels of fra-
grance ingredients based on induction and labeling of allergens.
In a previous opinion, the SCCP stated:
‘As atranol and chlorotranol are such potent allergens (and chloroatranol
particularly so), they should not be present in cosmetic products’.[48]
This opinion is largely based on results obtained from a bioassay-
guided fractionation of an oakmoss absolute.[1] From a method-
ological viewpoint, following a fractionation using an in vivo
allergy assay is a reductionistic approach, and one should be
aware that toxicity could derive from a synergistic interaction of
more than one compound, and that there could not be a single
‘culprit’ in case of a toxic activity. Most importantly, the SCCP
indicated that ‘oakmoss absolute is a strong sensitizer’, whereas
‘treemoss is unlikely to be a skin sensitizer under the conditions
of the test’ (local lymph node assay).[48] Hence, it would be logical
to take into account the toxicological tests performed on tree-
moss still containing high levels of 10 and 15 (see above), and
previous observations showing that ‘hydrolysed atranorin’ —thus
likely to contain 10 and/or haematommic acid 11—did not elicit
any reaction on three sensitized individuals out of eight who
had tested positive to atranorin, themselves representing 1% of
the tested patients.[49] Furthermore, one can question the validity
of results obtained from patch-testing sensitized humans with
the two synthetic atranols of uncertain purity, and from ‘high’ to
‘low’ concentrations.[50] Individuals who give low thresholds of
elicitation in sequential dilution patch tests are not always the
most responsive in repeated open application test (ROAT) studies,
and vice versa.[51]
An IFRA standard has recently been released that specifies that:
‘tree moss extracts shall not contain more than 0.8% of dehydroabietic acid
(DHA) as a marker of 2% of total resin acids. The concentration of DHA
(about 40% of the total resin acids) in tree moss can be measured with an
HPLC reverse phase-spectrofluorimetry method. Further, levels of atranol
and chloroatranol should each be below 100 ppm in tree moss extracts.’
Consequently, when ‘pine treemoss’ absolute oils with low risk
of ACD are desired in order to comply with existing recommenda-
tions and regulations, one needs to reduce the levels of both resin
acids and atranol/chloroatranol. This task appears to be quite
tricky, although it is not technically unattainable. In principle,
patented methods would indeed apply to both tasks.[45,52–54] The
only limiting factors are cost and odour effectiveness. Is this going
to trigger the disappearance of ‘pine treemoss’ from the market?
A recent publication reported that ‘oakmoss exerts prominent
photo toxicity in an in vitro assay with photohaemolysis occurring
upon exposure to both UVB- or UVA-rich sources’. Regretfully,
however, the exact nature and origin of the tested ‘oakmoss’
(true Evernia prunastri extract?) were not mentioned.[55]
Lichen Extracts as Fragrance Ingredients
In perfumery, lichen extracts are generally classified in the ‘woody’
and ‘green’ notes categories. The top note of true oakmoss abso-
lute provides a unique and characteristic seaweed and marine
effect that is highly prized by perfumers. Whether or not it is a
contribution of the algal portion of the symbiotic algae:fungus
of the lichen, this has sometimes prompted the addition to oak-
moss of seaweed extracts, in an attempt to enhance this effect
in a number of natural specialties. Phenolic notes are clearly
provided by volatile orcinol derivatives, and contribute to the
woody complexity of the product. While it is discrete but real in
the case of oak and cedar trees, the contribution of the host
plant is obvious in ‘pine treemoss’ extracts, with tar-like and heavy
pine needle/terpenic notes. In some cases, the plant material
is collected at ground level, together with true mosses or even
fungi, which are responsible for the earthy/mouldy character of
the extracts, reminiscent of humus and forest undergrowth.[56–58]
Undoubtedly these fragrance raw materials are unique and so
complex that they cannot be replaced by any synthetic blend.
The odour of lichen (so-called ‘moss’) extracts has not yet revealed
all of its secrets. Indeed, when carrying out extensive and
detailed fractionation of absolute oils, analytical chemists observe
that all fractions, irrespective of their complexity, still tend to smell
‘mossy’. This suggests that trace constituents play an important
organoleptic role.
Tab le 5. Quantitative analysis of treemoss absolutes pre-
pared from lichen growing on pine trees[13]
Component Regular
absolute
(%)
Resin
acid—less
absolute (%)
β
-Orcinol 7a0.016 0.01
Atranol 10a0.37 0.31
Methyl
β
-orcinolcarboxylate 9a3.70 3.55
Chloroatranol 15a0.22 0.15
β
-Orcinolcarboxylic acid 8a0.012 0.014
Methyl haematommate 12a0.20 0.20
Ethyl haematommate 13a0.22 0.97
Haematommic acid 11a0.075 0.044
Ethyl chlorohaematommate 17a0.4 0.4
Olivetonide 20a0.30 0.30
2′-O-methylphysodone 33a0.79 1.14
Physodone 32a2.39 4.69
β
-Sitosterol 60/Stigmasterol 63a4.12 8.0
Atranorin 25bND ND
Chloroatranorin 26bND ND
Dehydroabietic acidb,c 4.12 0.29
7-Oxodehydroabietic acidc0.19 0.09
aGC–MS after silylation with internal standardization. bHPLC
with external standardization. cGC–MS after methylation
(CH2N2) with internal standardization. ND, not detected (limit
of detection, ca. 30 p.p.m. in a resinoid).
Lichen extracts as raw materials in perfumery. Part 2: treemoss
Flavour Fragr. J. 2009, 24, 105–116 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/ffj
115
Conclusion
We have shown herein and in the previous review article that the
understanding of the chemistry of industrially relevant lichen
extracts—so-called ‘moss extracts’—is considerable. In the case
of treemoss, we now report more than 90 constituents. This should
put an end to allegations claiming that these complex products are
‘partly known’[50] in quoting irrelevant textbooks such as Arctander’s.[58]
On the other hand, it appears that so-called ‘moss’ resinoids
are manufactured by solvent extraction of a variety of lichens,
and sometimes of poorly defined mixtures of lichens. Whatever
the extraction technology, a serious concern persists about the
accurate naming of these important fragrance raw materials,
depending on the botanical species involved. As we have pointed
out several times, confusion has existed and still exists as to what
is really ‘oakmoss’ and ‘treemoss’. This confusion was aggravated
by the wide-spread allegation, albeit not always founded, that
cheaper treemoss was used to ‘cut’ (dilute) the more noble oak-
moss extracts. Undoubtedly, this confusion has been detrimental
to the fragrance industry in several respects. It would certainly
be desirable to redefine which product is which. Based on what
has been demonstrated in these articles, we now propose to
define only three categories of lichen (so-called ‘moss’) extracts:
• ‘Oakmoss’ extracts and their physically derived products,
which are obtained exclusively from Evernia prunastri, a
lichen that grows mainly, but not only, on oak trees.
• ‘Cedarmoss’ extracts, obtained exclusively from Pseudevernia
furfuracea growing on cedar trees (Cedrus atlantica).
• ‘Treemoss’ extracts, obtained from any other lichen species
growing on any host plant.
In principle, it would be easy to characterize such oakmoss resin-
oids or absolute oils by the specific presence of evernic acid and
evernin (or what remains of these depsides after processing),
along with large amounts of usnic acid, whereas cedarmoss resi-
noids would contain specifically physodic and/or isophysodic
acid but no usnic acid at all (<5 ppm; D. Joulain, unpublished
information).
Once these definitions are accepted and enforced, it would
be logical to carry out the appropriate toxicological tests, start-
ing from samples representative of industrial production, in
contrast with most experiments that have given rise to recent
publications dealing with the ACD of these fragrance raw
materials.
As we have mentioned above, the quantity of treemoss that is
processed today, for the manufacture of extracts for the fra-
grance industry, is only about one-third of what it was 10 years
ago. This is apparently the result of the industry’s self-regulation
with respect to resin acids. During the same period, the con-
sumption of oakmoss has also been declining, from 700 to 550
tons. Thus, the total consumption of lichens is now only 30% of
what it was in 1997. It is foreseeable that the new drastic con-
straints concerning the atranols will induce a further decrease in
the usage of lichen extracts for fragrance compounding.
It would therefore be desirable to undertake a comprehensive
survey of the uses of lichens, other than in fragrances, to up-date
the quantities of lichens that are really harvested and traded
in Europe and North America. This would possibly shed light on,
and reveal some causes of allergies induced by, otherwise unex-
pected exposure to these products.
Addendum
After the submission of the previous article, we became aware of
a publication reporting new constituents of Evernia prunastri
collected in the Canary Islands, Spain. Those that are missing
in the first article are listed in Table 6, and their structures are
shown in Figure 8.[59] It is interesting to note the identification
Tab le 6. New compounds identified in Evernia prunastri[59]
Compound no. Name CAS RN MW
85 Methyl everninate 520-43-4 196.2
86 Methyl di-O-methylorsellinate 6110-37-8 210.2
87 Methyl 2′-O-methyllecanorate 124521-14-8 346.3
88 Methyl 2′-O-methylevernate NA 360.4
89 Methyl 2,2′-di-O-methylevernate 79080-47-0 374.3
90 2,2′-Di-O-methylgyrophoric acid 863781-98-0 480.5
91 Prunastrin 863919-47-5 508.5
Figure 8. Chemical structures of new compounds identified in Evernia prunastri[59]
D. Joulain and R. Tabacchi
www.interscience.wiley.com/journal/ffj Copyright © 2009 John Wiley & Sons, Ltd. Flavour Fragr. J. 2009, 24, 105–116
116
of two new tridepsides 90 and 91. However, the identification
of several methyl esters raises the question of whether these
products exist as such in the lichen, or are formed during their
extraction with methanol.
Acknowledgements
We gratefully acknowledge Dr J. Boustie, Dr J. Elix, Dr S. Huneck
and P. Racine for their guidance and suggestions. We thank Mr. Guy
Robert and Mr. Christopher Sheldrake, perfumers, for their advice.
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