Báo cáo khoa học: Characterization of surface n -alkanes and fatty acids of the epiphytic lichen Xanthoria parietina, its photobiont a green alga Trebouxia sp., and its mycobiont, from the Jerusalem hills pot

Characterization of surface n -alkanes and fatty acids of the1 Department of Medicinal Chemistry and Natural Products, School of Pharmacy, PO Box 12065, The Hebrew University of Jerusale

Characterization of surface n -alkanes and fatty acids of the

1

Department of Medicinal Chemistry and Natural Products, School of Pharmacy, PO Box 12065, The Hebrew University of Jerusalem, 91120, Israel;2Division of Environmental Sciences, Graduate School of Applied Science, The Hebrew University of Jerusalem, Israel; 3 Department of Plant Pathology, ARO, Ministry of Agriculture, Bet Dagan, Israel

Surface alkanes and fatty acids from the thalli of the lichen

Xanthoria parietina, its photobiont Trebouxia sp., and its

mycobiont were analysed by GC-MS The green alga

(Z,Z,Z)-9,12,15-18 : 3 (Z,Z)-9,12-18 : 2 and (Z)-9-18 : 1,

However, the mycobiont contained mainly saturated fatty

acids such as hexadecanoic (16 : 0) and octadecanoic acid

Dehydroabietic acid was found in both lichen and

mycobi-ont The occurrence of different amounts of n-alkanes and fatty acids in the photobionts and mycobionts of X parietina was shown for the first time Lichens collected from different locations in the Jerusalem hills contained n-alkanes ranging

concentrations in the photobiont and mycobiont were 17–24

Trebouxia; Xanthoria

Lichens have been described as dual organisms because

they are symbiotic associations between two (or sometimes

more) entirely different types of micro-organism: a fungus

(termed the mycobiont) and a green alga or a

cyanobacte-rium (termed the photobiont) These organisms have both

algal and fungal properties [1,2] and produce n-alkane,

unusual betaine ether glycerolipids [3,4], and saturated,

unsaturated, branched, and halogenated fatty acids [5–20]

Many different bioactive secondary metabolites have also

been isolated from lichen species [21,22], which have been

used in pharmaceutical sciences [23] One of main questions

in lichen biology and chemistry is which compounds are

synthesized by which symbiotic lichen partner? As fungi are

often rich in secondary products [24,25,26], it is not surprising

that many typical lichen substances are synthesized by the

mycobiont Culberson et al [27] showed that experimentally

produced combinations of a mycobiont with foreign

photo-bionts generate the same lichen products as the mycobiont in

a natural thallus with its usual partner Fox & Huneck [28]

showed that mycobionts could synthesize long-chain

aliphatic acids, but they did not indicate which fatty acids were synthesized by the photobiont

In this study, we attempt to show which aliphatic hydrocarbons and fatty acids are produced by the photo-biont and which by the mycophoto-biont isolated from the lichen Xanthoria parietina

Materials and methods Lichen samples

Samples of X parietina lichens were collected from the bark

of trees in the Givat Ram Campus, Hebrew University, Abu Ghosh Village, Gilo Aleph and Ein Kerem (all on the

Jerusalem hills [29,30]

Isolation and cultivation of photobionts Photobiont was isolated by the micropipette method of Ahmadjian [31] and grown as described by Friedl [32] The photobiont was examined both in the lichenized and cultured state by standard light microscopic techniques with modifications as described by Dor [33] For identifi-cation, the isolated strains were compared with cultures of all known species of Trebouxia (Chlorophyta, Trebouxio-phyceae, Pleurastrophyceae) [34,35] The lichen photobiont

grown in the Laboratory of Hydrobiology Stock cultures of the alga were maintained on 3N Bold’s Basal Medium agar

Chemistry and Natural Products, School of Pharmacy, PO Box 12065,

The Hebrew University of Jerusalem, 91120, Israel.

Fax: + 972 2 675 8201, Tel.: + 972 2 675 7549,

E-mail: dvalery@cc.huji.ac.il

*Affiliated with The David R Bloom Centre for Pharmaceutical

Research, The Hebrew University of Jerusalem, Jerusalem 91120, Israel.

Note: Parts of this paper were presented at the 68th Israeli Chemist’s

Society, 27 January 2003, Tel Aviv.

(Received 28 November 2002, revised 6 February 2003,

accepted 5 March 2003)

0.025 gCaCl2; and 0.025 gNaCl In addition, 1 L medium

KOH, with improved conditions as described previously

[33] Hydrocarbons and fatty acids from fresh centrifuged

biomass (4.9 g) were extracted

Isolation and cultivation of mycobionts

Mycobionts from X parietina were obtained from the

spores discharged from apothecia of a thallus, and were

cultivated in test tubes containing20 gmalt extract (Difco),

months, the colonies and slants with crystals were harvested

The mycobionts used in our study were cultivated by J R

Extraction of hydrocarbons and fatty acids

Lichen thalli from X parietina and their photobiont

dichloromethane/methanol (40 : 30 : 30, by vol.) as

des-cribed previously [37]

GC–MS analysis

A Hewlett–Packard 5890 gas chromatograph (series II),

modified for a glass capillary column coupled to a HP

GC-mass selective detector (5971B MSD), was used

Hydrocar-bons and the methyl esters of fatty acids were analysed by GC

on two capillary coupled columns as described previously

[37], and also on an HP-5 column (10 m, internal diameter

0.32 mm, film thickness 0.25 mm) coupled with a second

capillary column RTX-1701 (Restek, Boca Raton,

0.25 lm film) The GC oven was programmed as follows:

70 eV The scan range was m/z 30–650, and scan rate 0.9

scans/second Solvent delay was set at 10 min

Hydro-carbons and fatty acid methyl esters were identified by

comparison with those found in the Wiley mass spectral

library (7th edition)

Results and discussion

The complex hydrocarbons and fatty acids produced by the

cultured photobiont green alga Trebouxia sp and the

mycobiont, and also by X parietina, were separated by

serially coupled capillary columns with consecutive

non-polar and seminon-polar stationary phases [37] GC-MS analysis

of the hydrocarbons and fatty acids of X parietina, its

photobiont and mycobiont collected from four different

locations indicated the presence of 27 n-alkanes and six

major fatty acids Quantitative data are shown in Table 1

The major constituents of lichen and mycobionts ranged

completely identified n-hydrocarbons The major n-alkanes

and fatty acid methyl esters identified from the lichen, photobiont and mycobiont is shown in Fig 1 Peak 5 [scan

299(18), 141(9), 197(8)] was found to be the methyl ester

of dehydroabietic acid This is the first time that dehydro-abietic acid has been found in both lichen and mycobiont This acid (of the triterpenoid resin family) which occurs widely in higher plants [38] is a typical constituent of coniferous resins and is highly reactive, participating in the protection of wounded trees against microbial attacks [39] Dehydroabietic acid has been found in traditional Chinese medicines, and is used in many Asian countries [40] It has also been found as a major effluent component in the paper and pulp industry [41] and is one of the toxic compounds accumulated in fish and other aquatic organisms [42] Ten major fatty acids were identified in the lichen, photobiont and mycobiont (Table 1) The mycobiont and lichen do not contain the (Z,Z,Z)-9,12,15-18 : 3 fatty acid, but this fatty acid is found in green alga only to a concentration of 9.8–17% (Fig 2) According to Bychek-Guschina [43], photobionts of Trebouxia erici and

St Petersburg, Russia) do not contain the 18 : 3 and 18 : 2 fatty acids, and produce only two fatty acids: 16 : 0 (30% and 51%, respectively) and 18 : 1 (69% and 42%, respect-ively) It is probably an artefact of the GC analysis Mycobionts produce saturated fatty acids such as tetra-decanoic acid (5–9%), hexatetra-decanoic acid (34–37%) and octadecanoic acid (23–30%) (Table 1) Three isomers, cis-9-18 : 1, cis-10-18 : 1 and cis-12-18 : 1, have been identified from the lichen, photobiont, and mycobiont

Lichens, symbiotic organisms of fungi and algae, syn-thesize numerous metabolites, lichen substances, which comprise aliphatic, cycloaliphatic, aromatic, and terpenic compounds Lichens and their metabolites have manifold biological activities such as antiviral, antibiotic, antitumor, allergenic, plant growth inhibitory, antiherbivore, and enzyme inhibitory [44–47] Usnic acid, a very active lichen substance, is used in pharmaceutical preparations [48] The leaves of higher plants contain waxy alkanes and have been shown to be useful for protection against UV-B radiation, as well as photoinhibition [49] Accordingto Piervittori et al [50], in X parietina, this layer is made up of

not show any statistical difference between the populations growing in the four different places (Table 1) On analysis of hydrocarbons from the lichen Lobaria pulmonaria [51] the

Argentinian lichens for the presence of alkanes, and found

1 ].

Lichen (192.31)

Photobiont (23.89)

Mycobiont (247.87)

Lichen (211.45)

Photobiont (17.28)

Mycobiont (262.12)

Lichen (206.52)

Photobiont (19.34)

Mycobiont (238.79)

Lichen (187.94)

Photobiont (21.67)

Mycobiont (215.16)

in E prunastri [54] These were the first reports of the

species Gaskell et al [55] reported hydrocarbons with chain

crispa, and Siphula ceratites Two collections of each lichen

were made several years apart but at the same locations The

S ceratites: 18 alkanes The fractions of alkanes from

spectrum that indicated it to be an equimolar mixture of

7-methyl and 8-methyl heptadecanes The same component

was found in trace amounts in the first sample of the two

hydro-carbons of the second sample of S ceratites were a series of

Twenty-one hydrocarbons were identified in Cetraria

(69.5%) Cultivated mycobionts isolated from lichens

collected in different places in the Jerusalem hills had the same hydrocarbon content as the lichen (Table 1) It is known that some lichen species contain both n-alkanes and branched alkanes [5], but our studies and others [50,53] have shown that X parietina contains only n-alkanes

More interestingexperimental data were obtained dur-ingstudies of the green alga Trebouxia sp It was surprising

to find that the major n-alkanes produced by the

light hydrocarbons have also been found as minor components in some lichen species, varyingfrom 0.12%

to 1.12% [51,52] GC separation of light hydrocarbons isolated from green alga Trebouxia sp is shown in Fig 1 This species is widespread in many lichens, including

belonging to this genus have been isolated from lichens; however, their hydrocarbons and fatty acids have not been analysed

Fig 1 Gas chromatographic separation on serially capillary coupled columns of n-alkanes and methyl esters of fatty acids from Xanthoria parietina (I), photobiont Trebouxia sp (II) and mycobiont (III) Lichen X parietina was collected in Givat Ram (Table 1) Major identified peaks show on three parts on GC-MS, peaks: 1, hexane-1,6-dioic acid, dimethyl ester; 2, tetradecanoic acid, methyl ester; 3, hexadecanoic acid, methyl ester;

4, octadecanoic acid, methyl ester; 5, dehydroabietic acid, methyl ester.

Successful separation of hydrocarbons and fatty acids

from the lichen, photobiont and mycobiont was achieved by

usingserially coupled capillary GC columns with

consecu-tive nonpolar and semipolar stationary phases as described

previously [37] We have also shown that the cultivated

photobiont Nostoc sp isolated from Collema sp lichen can

synthesize more than 130 metabolites including,

cyclo-hexane, cyclopentane, aliphatic saturated hydrocarbons

It has been shown that photobionts and mycobionts use

different biosynthetic pathways, for example, in the

biosyn-thesis of sterols Thus, Lenton et al [58] showed that the

photobionts Trebouxia sp and Trebouxia decolorans and

also the mycobiont isolated from X parietina synthesized

different sterols X parietina and its mycobiont were found

to contain ergosterol and lichesterol as the major

ergosta-7,22-dien-3b-ol, ergosta-7,24(28)-dien-3b-ol, and

ergosta-7,22,24(28)-trien-3b-ol The photobionts Trebouxia

sp and T decolorans contained predominantly

porifera-sterol, with lower levels of clioporifera-sterol, ergost-5-en-3b-ol, brassicasterol and cholesterol These results indicate the presence of separate biosynthetic pathways for some secondary metabolites in the photobionts and mycobionts

of lichens

Our results show that Trebouxia sp predominantly produces the followingunsaturated fatty acids:

9,12,15-18 : 3, 9,12-9,12,15-18 : 2, and 9-9,12,15-18 : 1, as well as lig ht n-alkanes

mycobiont are: tetradecanoic (14 : 0), hexadecanoic (16 : 0) and octadecanoic acid (18 : 0), and very long-chain

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